United States
Environmental Protection
Agency
EPA 821 4R-92-002
April 1992
Methods For The Determination
Of Nonconventional Pesticides
In Municipal And Industrial
Wastewater
I
I
I
0 2.0 4.0 6.0 8.0 10.0 12.0 14.0
Office Of Water
(WH-552)
1 ’ Printed on F ycIed P ip r
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United States Off ice of Water EPA/821/R/92/002
Environmental Protection Engineering and Analysis Division (WH-552) April 1992
Agency Washington. DC 20460
&EPA Methods for the Determination
of Nonconventional Pesticides in
Municipal and Industrial Wastewater
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Acknowledgements
This methods compendium was prepared under the direction of Thomas E. Fielding, Ph.D. and
William A. Telliard of EPA ’s Office of Water, Engineering and Analysis Division. This document was
prepared under EPA Contract No. 68-C9-0019 by Viar & Company, Environmental Services Division.
Interface Inc. and the Colorado State University Pesticide Study Center made significant contributions
to the underlying methods development research.
Disclaimer
This methods compendium has been reviewed by the Engineering and Analysis Division, U.S.
Environmental Protection Agency, and approved for publication. Mention of trade names or commercial
products does rot constitute endorsement or recommendation for use.
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Introduction
The Environmental Protection Agency (EPA) is proposing effluent limitations and guidelines at
40 CFR Part 455 to control the discharge of certain pesticide active ingredients into wastewater. This
compendium of test procedures (analytical methods) supports this proposal.
This compendium includes many methods for determination of active ingredients in wastewater
that have not previously been proposed or published, whether or not the active ingredient is included in
the proposal. The purpose of including all of these methods is to create a single reference for analysts
seeking to measure infrequently determined active ingredients.
These test procedures are proposed under the authority of Sections 304(h) and 501(a) of the
Federal Water Pollution Control Act (FWPCA) (33 U.S.C. 1251 et. seq.). These sections of the FWPCA
require EPA to promulgate guidelines establishing test procedures for the determination of pollutants in
wastewater.
Many of the test procedures in this compendium were listed in Appendix E of EPA’s original
promulgation of rules for the pesticides category (50 FR 40708). These test procedures were withdrawn
as a part of the remand of the pesticides rules in 1986 (51 FR 44911). Some of the test procedures that
appeared in the original promulgation have been updated to include more analytes and/or include
additional performance data.
Many of the test procedures in the original promulgation were also published by EPA’s Effluent
Guidelines Division in 1983 as publication EPA 4.40/1-83/079-C. This publication is now out of print.
The publication included industry methods, EPA developed methods, and contractor methods.
The test procedures in this compendium are methods developed by EPA’s Environmental
Monitoring Systems Laboratory in Cincinnati, Ohio, (EMSL-Ci), methods developed by EPA’s
Engineering and Analysis Division (EAD; formerly the Industrial Technology Division and the Effluent
Guidelines Division) within EPA’s Office of Science and Technology (formerly the Office of Water
Regulations and Standards), and an industry method for organo-tin compounds.
The EMSL-Ci methods were developed in the late 1970s and early 1980s. Some have been
updated in the interim. These methods have three digit numbers beginning with 6 (e.g., 622). The
methods written by EM) were developed to measure active ingredients in support of the pesticides
rulemaking, and have therefore been applied to the specific wastewater for which they were intended.
These methods have four digit numbers beginning with 16 (e.g., 1656). The industry methods for
organo-tin are Methods EV-024 and EV-025.
This compendium comprises 41 different EPA methods and one industry method. A summary
of all the analytes that may be detected using these methods is shown in in the Cross Reference table.
Some analytes can be detected by more than one method.
In addition to methods developed for today’s proposed rule, EPA is investigating other methods
and other analytical techniques to aid in the determination of non-conventional pesticides and other
analytes of concern. EPA is interested in simplifying methods, where possible, and in reducing the
potential pollution threat caused by the volumes of solvents used in some methods. An example of a
simplification technique is the use of an iinmunoassay specific to a given analyte (such as a pesticide) or
analyte group (such as the phenoxyacid herbicides) to allow EPA to screen rapidly for these analytes in
discharges and in other environmental samples. EPA is also investigating the use of “solid phase
extraction” (liquid-solid extraction) as a means of reducing the amount of solvent used in conventional
1
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extraction procedures. Solid phase extraction (SPE) has been successfully applied to drinking water
mali Ices, but initial tests with wastewaters containing high dissolved solids yielded low recoveries of the
analytes of concern. More recent materials have yielded recoveries more consistent with conventional
extraction techniques. EPA will continue to investigate these and other analytical techniques with the
objective of producing lower cost, more rapid, and less environmentally damaging analytical methods.
Questions about the content of this document should be directed to:
W. A. Telliard
U.S. EPA (WH-552)
Office of Science and Technology
Engineering and Analysis Division
401 M Street, SW
Washington, DC 20460
(202) 260-7131
or
U.S. EPA Sample Control Center
Viar & Company
P.O. Box 1407
Alexandria, VA 22313
(703) 557-5040
U
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Contents
Method #
604.1
608.1
608.2
614
614.1
615
616
617
618
619
620
622
622.1
627
629
630
630.1
631
632
632.1
633
633.1
634
635
636
637
638
639
640
641
642
643
644
645
646
1656
1657
1658
1659
1660
1661
Parameters
Hexachiorophene and Dichiorophen
Organochiorine Pesticides
Certain Organochiorine Pesticides
Organophosphorus Pesticides
Organophosphorus Pesticides
Chlorinated Herbicides
Certain Carbon-, Hydrogen-, and Oxygen-Containing
Organohalide Pesticides and PCBs
Volatile Pesticides
Triazine Pesticides
Diphenylamine
Organophosphorus Pesticides
Thiophosphate Pesticides
Dinitroaniline Pesticides
Cyanazine
Dithiocarbamate Pesticides
Dithiocarbamate Pesticides
Benomyl and Carbendazim
Carbamate and Urea Pesticides
Carbamate and Amide Pesticides
Organonitrogen Pesticides
Neutral Nitrogen-Containing Pesticides
Thiocarbate Pesticides
Rotenone
Bensulide
MBTS and TCMTB
Oryzalin
Bendiocarb
Mercaptobenzothiazole
Thiabendazole
Biphenyl and Ortho-Phenyiphenol
Bentazon
Picloram
Certain Amine Pesticides and Lethane
Dinitro Aromatic Pesticides
Organohalide Pesticides
Organophosphorus Pesticides
Phenoxy-Acid Herbicides
Dazomet
Pyrethrins and Pyrethroids
Bromoxynil
____ Pesticides
Page
.1
19
39
59
79
97
117
137
169
187
209
229
247
269
287
303
317
331
347
365
379
397
417
437
455
471
489
507
525
543
557
571
585
601
619
635
679
717
753
769
789
III
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Contents (can. )
Appendix
Methods EV-024 and EV-025:
Analytical Procedures for Determining Total Tin and Triorganot in Wastewater 805
Iv
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Cross-Reference
Parameter
GAS No.
Applicable Method(s)
Acephate
Acifluorfen
Alachior
Aldrm
Allethrin (pynamin)..
Ame yn
Aminocarb
Amobam
AOP
Aspon
Atraton
Atrazine
Azinphos ethyl
Azinphos methyl
Barban
Basalin (Fluchioralin)
Bendiocarb
Benfluraim
Benomyl
Bensulide
Bentazon (Basagran)
a-BHC
fi-BHC
y-BHC
-BHC
Biphenyl
Bromadil
Bromoxynil octanoate
Bromoxynil
Busan40
Busan85
Butachior
Butylate
Captafol
Captan
Carbam-S
Carbaryl
30560-19-1
50594-66-6
15972-60-8
309-00-2
584-79-2
834-12-8
2032-59-9
3566-10-7
3244-90-4
1610-17-9
1912-24-9
2642-71-9
86-50-0
101-27-9
33245-39-5
22781-23-3
1861-40-1
17804-35-2
741-58-2
25057-89-0
319-84-6
319-85-7
58-89-9
319-86-8
92-52-4
314-40-9
1689-99-2
1689-84-5
51026-28-9
128-03-0
23184-66-9
2008-41-5
2425-06-1
133-06-2
128-04-1
63-25-2
1656, 1657
1656
645
617, 1656
1660
619
632
• . . 630, 630.1
630
622.1
619
• . . . 619, 1656
1657
614, 622, 1657
632
646
639
• . . . 627, 1656
631
636
643
617, 1656
• . . 617, 1656
• . . 617, 1656
• . . 617, 1656
642, 1625
• . . . 633, 1656
1656
• . 1661, 1625
630, 630.1
630, 630.1
• . . 645, 1656
634
1656
617, 1656
• . 630, 630.11
632
‘Carbain-S was not explicitly listed in these methods, but these methods are applicable to
pesticides such as Carbam-S.
V
dithiocarbamate
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Cross-Reference (c t.)
Parameter
CASNo.
Applicable Method(s)
Carbezx laziin
10605-21-7
Carbofuran
1563-66-2
Carbophenothion
786-19-6
CDN
97-00-7
Ch lordane
5103-74-2
Oilorfeviuphos
adoroben_ziiate
470-90-6
510-15-6
Chioroneb
2675-77-6
Chioropicrin
76-06-2
Chioropropylate
5836-10-2
adorothalonil
1897-45-6
101-21-3
ChlOfpyrifos methyl
5598-13-0
Chlorpyrilbs
2921-88-2
Cotimaphos
56-72-4
Crotoxyphos
7700-17-6
Cyanazine
21725-46-2
•
1 134—23—2
C3rcloprate
5446O 46 —7
Cylhithrin Oaytbxoid)
68359-37-5
Daispon
75-99 -0
Da ind
533-744
2,4—D
94—75—7
2,4—DB
94—826
DBCP
96-12-8
D PA
1861-32-1
4,4’-DDD
4,4’-DDE .
72—55—9
4,4’ -DDT
50-29-3
Deet
134-62-3
DEF
D neton
806548-3
DiaJ1 e
2303164
Diarinon
I)ibsoncb lornprnsttt le
333415
96128
Dicamba
D ldilcfeiwhion
D1chloi e
1918-00-9
97-17-6
1 17—80—6
Dlcbloraii .
99—30—9
Dichioroplien
Dichiorprop
9723-4
120-36-5
615, 1658
631
632
617, 1656
646
617, 1656
1657
608.1, 1656
• . . . 608.1, 1656
618
• . .. 608.1, 1656
608.2, 1656
632
622, 1657
622, 1657
622, 1657
1657
629
634
616
1660
615, 1658
1659
615, 1658
615, 1658
1656
608.2
617, 1656
617, 1656
617, 1656
633
1651
614, 622, 1657
1656
614, 622, 1657
608.1
615, 1658
- . . . 622.1, 1657
1656
608.2,617
604.1
VI
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Cross-Reference (cont.)
Parameter
GAS No.
Applicable Method(s)
Hexachiorophene
70-30-4.
604.1
Dichiorvos
62-73-7
622, 1657
Dicofol
115-32-2
617, 1656
Dicrotophos
141-66-2
1657
Dieidrm
60-57-1
617, 1656
Dimethoate
60-51-5
1657
Dinocap
Dinoseb
39300-45-3
88-85-7
646
615, 1658
Dioxathion
78-34-2
614.1, 1657
Diphenamid
Diphenylamine
Disulfoton
957-51-7
122-39-4
298-04-4
645
620
614, 622, 1657
Diuron
330-54-1
632
Endosulfan I
959-98-8
617, 1656
Endosulfanil
33213-65-9
617, 1656
Endosulfan sulfate
1031-07-8
617, 1656
Endrin aldehyde
Endrin
7421-93-4
72-20-8
617, 1656
617, 1656
Endrin ketone
53494-70-5
1656
EPN
2104-64-5
614.1, 1657
Evrc
759 .944
634
Ethalfiuralin
55283-68-6
627, 1656
Ethion
563-12-2
614,614.1, 1657
Ethoprop
Ethylene dibromide
Etridiazole
13194-48-4
106-93-4
2593-15-9
622, 1657
618
608.1, 1656
xD
502-55-6
630.1
Famphur
Fenarimol
52-85-7
60 168-88-9
622.1, 1657
633.1, 1656
Fenitrothion
122-14-5
622.1
Fensulfothion
115-90-2
622, 1657
Fenthion
55-38-9
622, 1657
Fenuron
101-42-8
632
Fenuron-TCA
4482-55-7
632
Fenvalerate
51630-58-1
1660
Ferbam
14484-64-1
630, 630.1
Fluometuron
2164-17-2
632
Fiuridone
59756-60-4
645
Fonophos
Heptachior epoxide
Heptachior
944-22-9
1024-57-3
76-44-8.. .
622.1
617, 1656
617, 1656
V I ’
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Cross-Reference (coin.)
Parameter
CASN0.
Applicable Method(s)
}Iexam by1-
phosphoramide
Merphos
Mefh m
Me*hanñdophos
Methiocarb
Methomyl
Methoprene
Methoxychior
Metribuzin
MevInplios
Mexacarbate
MGK264-A
MGK 264-B
MGK326
fwex
Molinate
Monuron
Monuron-TCA.
Nabam
Nabonate
Naled
Napropamide
Neburon
1 -lexazinone
1sod n....
Isopropalin.
Kepone
K .
KN Methyl.
Leptophos..
Lethane
Linuron
Malathion..
Mancozeb..
Maneb ..
MB
MGPA....
MCPP
680-31-9 1657
51235-04-2 633
465-73-6 617, 1656
33820-53-0 627, 1656
143-50-0 1656
42588-37-4 616
137 -41-7 630, 630.1
21609-90-5 1657
112-56-1 645
330-55-2 632
121-75-5 614, 1657
8018-01-7 630
12427-38-2 630
120-78-5 637
94-74-6 615,1658
7085-19-0 615, 1658
149-304
iso-so-s 622, 1657
137-42-8 630, 630.1
10265-92-6 1657
2032-65-7 632
16752-77-5 632
40596-69-8 616
72-43-5 608.2, 617, 1656
21087-64-9 633, 1656
776-34-7 622, 1657
315-18-4 632
113-48 -4 633.1
113-484 633.1
13645-8 633.1
2385-85-5 617, 1656
2212-67-1 634
6923 224 1657
150-68-5 632
140-41-0 632
142-59-6 630,630.1
138-93-2 630.1
300-76-5 622, 1657
15299 -99-7 632.1
555..37.3 632
yin
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Cross-Reference (cont.)
Parameter
CASNo.
Applicable Method(s)
Niacide
Nitrofen (TOK)
Norfiorazon
Organo-tin
Oryzaim
Oxamyl
Parathion ethyl
Parathion methyl
PCB-1016....
PCB-1221
PCB-1232 .
PCB- 1242
PCB- 1248.
PCB-1254.
PCB-1260....
PCNB
Pebulate
Pendimethalin
Pennethrin
Perthane
o-Phenylpheno l
Phorate
Phosmet
Phosphaniidon
Picloram
Polyram
Profluralin
Prometon
Prometryn
Pronamide
Propachior
Propanil
Propazine
Propham
Propoxur .
Pyrethrin I
Pyrethrin IL
Resmethrin
Ronnel
Ronnel
638
632
1657
1657
1656
1656
1656
1656
1656
1656
1656
1656
634
1656
1660
1656
642
8011-66-3 630
1836-75-5 1656
27314-13-2 645, 1656
EV24 and EVM25
19044-88-3
23135-22-0
56-38-2 614,
298-00-0 614, 622,
12674-11-2 617,
11104-28-2 617,
11141-16-5 617,
53469-21-9 617,
12672-29-6 617,
11097-69-1 617,
11096-82-5 617,
82-68-8 608.1, 617,
1114-71-2
40487-42-1
52645-53-1 608.2, 1656,
72-56-0 617,
132-27-4
298-02-2
732-11-6
13171-21-6
1918-02-1
9006-42-2
26399-36-0
1610-18-0
7287-19-6
23950-58-5
1918-16-7
709-98-8
139-40-2
122-42-9
114-26-1
121-21-1
121-29-9
10453-86-8
299-84-3
299-84-3
• . . 622, 1657
o . 622.1, 1657
1657
644
630
627
619
619
633.1
608.1, 1656
632.1
619, 1656
632
632
1660
1660
616, 1660
1657
622, 1657
Rotenone
83-79-4.
635
ix
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Cross-Reference cont.
Parameter
CAS No.
Applicable Method(s)
2625945-0
1982-49-6
122-34-9
1014-70-6
128-04-1
961-11-5 . -
8001-50-1
3689-24-5
35400-43-2
26002-80-2
1918-18-9
93-76-5..
21564-17-0
107-49-3
5902-51-2 *
13071-79-9
5915-41-3
886-50-0
7696-12-0
148-79-8
297-97-2
137-26-8
34643-46-4
8001-35-2
93-fl-i ... -
43 12143-3 -
52-458-6....
327-98-0
78-30-8...
41814-78-2
1582-09-8
512-56-1
953-17-3
53558-25-1
1929-77-7
12122-67-7
137-30-4
619
632
619, 1656
619
630, 630.1
622, 1657
617, 1656
1657
622
1660
632
615,1658
637
1657
633, 1656
614.1, 1657
619,1656
619
1660
641
622.1
630, 630.1
622, 1657
617, 1656
615, 1658
633, 1656
1657
622,1657
1657
633
617, 627, 1656
1657
1657
632.1
634
630
630, 630.1
630,630.1
Secbumeton
Siduron
Simazine
Simetryn
Sodium dimethyldithiocarbamate
Stiroibs (Tetrachlorvinphos)
Strobane
Sulfotepp
Suiprofos (boistar)
Sumithrin (phenothrm)
Swep.
24,5-T
TCMTh
TEPP
Te bacil
TeIbUfOS
Terbuthylazine
Tetbutryn
Tetramethrin.
Thiabendazole
Thionazin
Thram
Tokuthion
Toxaphene
2,4,5-TP
Triadimefon
Trichiorofon
Trichioronate.
Tricresyiphosphate
Tricyclazole
Trifluralin
Trimethyiphosphate
Trithion methyl
Vacor
Vernolate
ZAC
Zineb
Ziram
x
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Method 604.1
The Determination of
Hexachiorophene and
Dichiorophen in Municipal and
Industrial Waste waters
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Method 604.1
The Determination of Hexachiorophene and Dichiorophen in
Municipal and Industrial Wastewater Method
1. SCOPE AND APPLICATION
1.1 This method covers the determination of certain phenolic pesticides. The following parameters
can be determined by this method:
Parameters CASNo.
Dichlorophen 97-23-4
Hexachiorophene 70-30-4
1.2 This is a high performance liquid chromatographic (HPLC) method applicable to the determination
of the compounds listed above in industrial and municipal discharges as provided under
40 CFR 136.1. Any modification of this method beyond those expressly permitted shall be
considered a major modification subject to application and approval of alternative test procedures
under 40 CFR 136.4 and 136.5.
1.3 The method detection limit (MDL, defined in Section 14) for each compound is listed in Table 1.
The MDL for a specific wastewater may differ from those listed, depending upon the nature of
interferences in the sample matrix.
1 .4 This method is restricted to use by or under the supervision of analysts experienced in the use of
liquid chromatography and in the interpretation of liquid chromatograms. Each analyst must
demonstrate the ability to generate acceptable results with this method using the procedure
described in Section 8.2.
1.5 When this method is used to analyze unfamiliar samples for any or all of the compounds above,
compound identifications should be supported by at least one additional qualitative technique.
This method describes analytical conditions for a second liquid chromatographic column that can
be used to confirm measurements made with the primary column.
2. SUMMARY OF METHOD
2.1 A measured volume of sample, approximately 1 L, is adjusted to pH 4 to 4.5 and extracted with
methylene chloride using a separatory funnel. The methylene chloride extract is dried and
exchanged to methanol during concentration to a volume of S mL or less. Liquid
chromatographic conditions are described which permit the separation and measurement of the
compounds in the extract by HPLC using an ultraviolet detector (UVD). 1
3. INTERFERENCES
3.1 Method interferences may be caused by contaminants in solvents, reagents, glassware, and other
sample-processing apparatus that lead to discrete artifacts or elevated baselines in liquid
chromatograms. All reagents and apparatus must be routinely demonstrated to be free from
3
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Method 604.1
interferences under the conditions of the analysis by running laboratory reagent blanks as
described in Section 8.5.
3.1.1 Glassware must be scrupulously cleaned. 2 Clean all glassware as soon as possible after
use by rinsing with the last solvent used in it. Follow by washing with hot water and
detergent and thorough rinsing with tap and reagent water. Drain dry, and heat in an
oven or muffle furnace at 400°C for 15 to 30 minutes. Do not heat volumetric ware.
Some thermally stable materials, such as PCBs, may not be eliminated by this treatment.
Thorough rinsing with acetone aix! pesticide-quality hexane may be substituted for the
heating. After drying and cooling, seal and store glassware in a clean environment to
prevent any accumulation of dust or other contaminants. Store inverted or capped with
aluminum foil.
3.1.2 The use of high-purity reagents and solvents helps to minimize interference problems.
Purification of solvents by distillation in all-glass systems may be required.
3.2 Matrix interferences may be caused by contaminants that are coextractad from the sample. The
extent of matrix interferences will vary considerably from source to source, depending upon the
nature and diversity of the industrial complex or municipality being sampled. The acid/base
extraction cleanup described in Section 10 can be used to overcome many of these interferences,
but unique samples may require additional cleanup approaches to achieve the MDL listed in
Table 1.
4. SAFETY
4.1 The toxicity or carcinogenicity of each reagent used in this method has not been precisely defined;
however, each chemical compound should be treated as a potential health hazard. From this
viewpoint, exposure to these chemicals must be reduced to the lowest possible level by whatever
means available. The laboratory is responsible for maintaining a current awareness file of OSHA
regulations regarding the safe handling of the chemicals specified in this method. A reference file
of material data handling sheets should also be made available to all personnel involved in the
chemical analysis. Additional references to laboratory safety are available and have been
identified’ for the information of the analyst.
5. APPARATUS AND MATERIALS
5.1 Sampling equipment, for discrete or composite sampling.
5.1.1 Grab-sample bottle: Amber borosilicate or flint glass, 1-L or i-quart volume, fitted with
screw-caps lined with PTFE. Aluminum foil may he substituted for PiPE if the sample
is not corrosive. If amber bottles are not available, protect samples from light. The
container and cap liner must be washed, rinsed with acetone or methylene chloride, and
dried before use to minimize contamination.
5.1.2 Automatic sampler (optional): Must incorporate glass sample containers for the
collection of a minimum of 250 mL. Sample containers must be kqfl refrigerated at 4°C
and protected from hgbt during compositing. If the sampler uses a peristaltic pump, a
minimum length of compressible silicone rubber tubing may be used Before use,
however, the compressible tubing should be thoroughly rinsed with methanol, followed
by repeated rinsings with distilled water to minimize the potential for contamination of
4
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Method 604.1
the sample. An integrating flow meter is required to collect flow-proportional
composites.
5.2 Glassware. (All specifications are suggested. Catalog numbers are included for illustration only.)
5.2.1 Separatory funnel: 2000-mL, with PTFE stopcock.
5.2.2 Drying column: Chromatographic column 400 mm long by 10 mm ID with coarse fit.
5.2.3 Chromatographic column: 400 mm long by 19 mm ID with 250-mL reservoir at the top
and PTFE stopcock (Kontes K-420290 or equivalent).
5.2.4 Concentrator tube, Kuderna-Danish: 25-rnL, graduated (Kontes K-570050-0250 or
equivalent). Calibration must be checked at the volumes employed in the test. A
ground-glass stopper is used to prevent evaporation of extracts.
5.2.5 Evaporative flask, Kuderna-Danish: 1000-mL (Kontes K-570001-1000 or equivalent).
Attach to concentrator tube with springs.
5.2.6 Snyder column, Kuderna-Danish: Three-ball macro (Kontes K-503000-0121 or
equivalent).
5.2.7 Snyder column, Kuderna-Danish: Two-ball micro (Kontes K-569001-0219 or
equivalent).
5.2.8 Vials: Amber glass, 5- to 10-mL capacity with PTFE-lined screw-cap.
5.3 Boiling chips: Approximately 10/40 mesh carborundum. Heat to 400°C for 4 hours or perfrom
a Soxhiet extraction with methylene chloride.
5.4 Water bath: Heated, capable of temperature control (±2°C). The bath shouLd be used in a hood.
5.5 Balance: Analytical, capable of accurately weighing to the nearest 0.0001 g.
5.6 Liquid chromatograph: Analytical system complete with liquid chromatograph and all required
accessories including syringes, analytical columns, detector, and strip-chart recorder. A data
system is recommended for measuring peak areas.
5.6.1 Pump: Isocratic pumping system, constant flow.
5.6.2 Column 1: Reversed-phase column, 5 ,L Spherisorb-ODS, 250 mm long by 4.6 mm or
equivalent. This column was used to develop the method performance statements in
Section 14. Alternative columns may be used in accordance with provisions described
in Section 12.1.
5.6.3 Column 2: Reversed-phase column, 5 Lichrosorb RP-2, 250 mm long by 4.6 mm or
equivalent.
5.6.4 Detector: Ultraviolet absorbance detector, 254 nm. This detector has proven effective
in the analysis of wastewaters for the parameters listed in the scope and was used to
develop the method performance statements in Section 14. Alternative detectors may be
used in accordance with the provisions described in Section 12.1.
6. REAGENTS
6.1 Reagent water: Reagent water is defined as a water in which an interferent is not observed at the
method detection limit of each parameter of interest.
6.2 Methylene chloride, methanol, acetonitrile: Distilled-in-glass quality or equivalent.
5
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Method 604.1
6.3 Sodium sulfate: ACS, granular, anhydrous. Heat in a muffle furnace at 400°C overnight.
6.4 Sodium phosphate, monobasic: ACS, crystal.
6.5 iN sodIum hydroxide: Dissolve 4.0 grams of NaOH (ACS) in 100 mL of distilled water.
6.6 Phosphoric acid (85%).
6.7 Stock standard solutions (1.00 1 zgf L): Stock standard solutions can be prepared from pure
standard materials or purchased as certified solutions.
6.7.1 Prepare stock standard solutions by accurately weighing about 0.0 100 g of pure material.
Dissolve the material in distilled-in-glass quality methanol and dilute to volume in a
10-mi.. volumetric flask. Larger volumes can be used at the convenience of the analyst.
If compound purity is certified at 96% or greater, the weight can be used without
correction to calculate the concentration of the stock standard. Commercially- prepared
stock standards can be used at any concentration if they are certified by the manufacturer
or by an independent source.
6.7.2 Transfer the stock standard solutions into PTFE-sealed screw-cap bottles. Store at 4°C
and protect from light. Frequently check stock standard solutions for signs of
degradation or evaporation, especially just prior to preparing calibration standards from
them,
6.7.3 Stock standard solutions must be replaced after 6 months, or sooner if comparison with
check standards indicates a problem.
7. CAUBR4 WON
7.1 Establish liquid chromatographic operating parameters equivalent to those indicated in Table 1.
The liquid chromatographic system can be calibrated using either the external standard technique
(Section 7.2) or the internal standard technique (Section 7.3).
7.2 External standard calibration procedure:
7.2.1 For each parameter of interest, prepare calibration standards at a minimum of three
concentration levels by adding accurately measured volumes of one or more stock
standards to a volumetric flask and diluting to volume with 50/50 methanol/water. One
of the external standards should be representative of a concentration near, but above, the
method detection limit. The other concentrations should correspond to the range of
concentrations expected in the sample concentrates or should define the working range
of the detector.
7.2.2 Using injections of 20 to 50 L of each calibration standard, tabulate peak height or area
responses against the mass injected. The results can be used to prepare a calibration
curve fbi each parameter. Alternatively, the ratio of the response to the mass injected,
defined as the calibration factor (CF), may be calculated for each parameter at each
standard concentration. If the relative standard deviation of the calibration factor is less
than 10% over the working range, the average calibration factor can be used in place of
a calibration curve.
7.2.3 The working calibration curve or calibration factor must be verified on each working
shift by the measurement of one or more calibration standards. If the response for any
compound varies from the predicted response by more than ± 10%, the test must be
6
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Method 604.1
repeated using a fresh calibration standard. Alternatively, a new calibration curve or
calibration factor must be prepared for that parameter.
7.3 Internal standard calibration procedure: To use this approach, the analyst must select one or more
internal standards similar in analytical behavior to the compounds of interest. The analyst must
further demonstrate that the measurement of the internal standard is not affected by method or
matrix interferences. Due to these limitations, no internal standard applicable to all samples can
be suggested.
7.3.1 Prepare calibration standards at a minimum of three concentration levels for each
parameter of interest by adding volumes of one or more stock standards to a volumetric
flask. To each calibration standard, add a known constant amount of one or more
internal standards, and dilute to volume with 50/50 methanol/water. One of the standards
should be a representative of a concentration near, but above, the method detection limit.
The other concentrations should correspond to the range of concentrations expected in
the sample concentrates, or should define the working range of the detector.
7.3.2 Using injections of 20 to 50 L of each calibration standard, tabulate the peak height or
area responses against the concentration for each compound and internal standard.
Calculate response factors (RF) for each compound as follows:
Equation 1
RF = (A,)(C 1, )
(4,) (C,)
.
where
A, =
4, =
C, =
C, =
Response for the parameter to be measured
Response for the internal standard
Concentration of the internal standard, in igIL
Concentration of the parameter to be measured, in g/L
If the RF value over the working range is constant, less than 10% relative standard
deviation, the RF can be assumed to be invariant and the average RF can be used for
calculations. Alternatively, the results can be used to plot a calibration curve of response
ratios, A/Ai , against RF.
7.3.3 The working calibration curve or RF must be verified on each working shift by the
measurement of one or more calibration standards. If the response for any compound
varies from the predicted response by more than ±10%, the test must be repeated using
a fresh calibration standard. Alternatively, a new calibration curve must be prepared for
that compound.
7.4 Before using any cleanup procedure, the analyst must process a series of calibration standards
through the procedure to validate elution patterns and the absence of interferences from the
reagents.
7
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Method 604.7
8.. QuAliTY CONTROL
8.1 Each laboratory using this method is required to operate a formal quality control program. The
minimum requirements of this program consist of an initial demonstration of laboratory capability
and the analysis of spiked samples as a continuing check on performance. The laboratory is
required to maintain performance records to define the quality of data that is generated.
81.1 Before performing any analyses, the analyst must demonstrate the ability to generate
acceptable accuracy and precision with this method. This ability is established as
described in Section 8.2.
8.1.2 In recognition of the rapid advances occurring in chromatography, the analyst is
permitted certain options to improve the separations or lower the cost of measurements.
Each time such modifications to the method are made, the analyst is required to repeat
the procedure in Section 8.2.
8.1.3 The laboratory must spike and analyze a minimum of 10% of all samples to monitor
continuing laboratory perfomance. This procedure is described in Section 8.4.
8.2 To establish the ability to generate acceptable accuracy and precision, the analyst must perform
the following operations.
8.2.1 Select a representative spike concentration for each compound to be measured. Using
stock standards, prepare a quality control check sample concentrate in methanol, 1000
times more concentrated than the selected concentrations.
8.2.2 Using a pipette, add 1.00 mL of the check sample concentrate to each of a minimum of
four 1000-mL aliquots of reagent water. A representative wastewater may be used in
place of the reagent water, but one or more additional aliquots must be analyzed to
determine background levels, and the spike level must exceed twice the background level
for the test to be valid. Analyze the aliquots according to the method beginning in
Section 10.
8.2.3 Calculate the average percent recovery (R), and the standard deviation of the percent
recovery (s), for the results. Wastewater background corrections must be made before
R and s calculations are performed.
8.2.4 Using the appropriate data from Table 2, determine the recovery and single-operator
precision expected for the method, and compare these results to the values measured in
Section 8.2.3. If the data are not comparable, the analyst must review potential problem
areas and repeat the test.
8.3 The analyst must calculate method performance criteria and define the performance of the
laboratory for each spike concentration and parameter being measured.
8.3.1 Calculate upper and lower control limits for method performance as follows:
Upper Control Limit (UCL) = R + 3s
Lower Control Limit (LCL) = R - 3s
where R and s are calculated as in Section 8.2.3. The UCL and LCL can be used to
construct control charts 6 that are useful in observing trends in performance.
8
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Method 604.1
8.3.2 The laboratory must develop and maintain separate accuracy statements of laboratory
performance for wastewater samples. An accuracy statement for the method is defined
as R ± s. The accuracy statement should be developed by the analysis of four aliquots
of wastewater as described in Section 8.2.2, followed by the calculation of R and s.
Alternatively, the analyst may use four wastewater data points gathered through the
requirement for continuing quality control in Section 8.4. The accuracy statements
should be updated regularly. 6
8.4 The laboratory is required to collect in duplicate a portion of their samples to monitor spike
recoveries. The frequency of spiked sample analysis must be at least 10% of all samples or one
sample per month, whichever is greater. One aliquót of the sample must be spiked and analyzed
as described in Section 8.2. If the recovery for a particular compound does not fall within the
control limits for method performance, the results reported for that compound in all samples
processed as part of the same set must be qualified as described in Section 13.3. The laboratory
should monitor the frequency of data so qualified to ensure that it remains at or below 5%.
8.5 Before processing any samples, the analyst should demonstrate through the analysis of a 1-L
aliquot of reagent water that all glassware and reagent interferences are under control. Each time
a set of samples is extracted or there is a change in reagents, a laboratory reagent blank should
be processed as a safeguard against laboratory contamination.
8.6 It is recommended that the laboratory adopt additional quality assurance practices for use with this
method. The specific practices that are most productive depend upon the needs of the laboratory
and the nature of the samples. Field duplicates may be analyzed to monitor the precision of the
sampling technique. When doubt exists over the identification of a peak on the chromatogram,
confirmatory techniques, such as liquid chromatography with a dissimilar column, must be used.
Whenever possible, the laboratory should perform analysis of standard reference materials and
participate in relevant performance evaluation studies.
9. SAMPLES COLLECTION, PRESERVATION, AND HANDLING
9.1 Grab samples must be collected in glass containers. Conventional sampling practices 7 should be
followed; however, the bottle must not be prerinsed with sample before collection. Composite
samples should be collected in refrigerated glass containers in accordance with the requirenents
of the program. Automatic sampling equipment must be as free as possible of plastic and other
potential sources of contamination.
9.2 The samples must be iced or refrigerated at 4°C from the time of collection until extraction.
9.3 Adjust the pH of the sample to 6 to 8 with sodium hydroxide or sulfuric acid immediately after
sampling.
10. SAMPLE EXTRACTION
10.1 Mark the water meniscus on the side of the sample bottle for later determination of sample
volume. Pour the entire sample into a 2-L separatory funnel.
10.2 The analyst may solvent-wash the sample at basic pH as described in Sections 10.2.1 and 10.2.2
to remove potential method interferences. For relatively clean samples, the wash should be
omitted and the extraction, beginning with Section 10.3, should be followed.
9
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Method 604.1
10.2.1 Adjust the pH of the sample to 12.0 with iN sodium hydroxide.
10.2.2 Add 60 mL of methylene chloride to the separatory funnel and extract the sample by
shaking the funnel for 2 minutes with periodic venting to release excess pressure. Allow
the organic layer to separate from the water phase for a minimum of 10 minutes. If the
emulsion interface between layers is more than one-third the volume of the solvent layer,
the analyst must employ mechanical techniques to complete the phase separation. The
optimum technique depends upon the sample, but may include stirring, filtration of the
emulsion through glass wool, centrifugation, or other physical methods. Discard the
methylene chloride extract. Perform a second and third extraction in the same maimer.
10.3 Add 50 g of NaH 2 PO 4 to the sample in the separatory funnel and shake to dissolve the solid. The
sample pH should be between 4.0 and 4.5. If necessary, adjust the pH with phosphoric acid or
sodium hydroxide. Add 200 mL of methylene chloride to the separatory funnel and extract the
sample by shaking the funnel for 2 minutes with periodic venting to release excess pressure.
Allow the organic layer to separate from the water phase for a minimum of 10 minutes. If the
emulsion interface between layers is more than one-third the volume of the solvent layer, the
analyst must employ mechanical techniques to complete the phase separations. Collect the
methylene chloride extract in a 1-L Erlenmayer flask.
10.4 Add a second 200 mL volume of methylene chloride to the separatory funnel and repeat the
extraction procedure a second time combining the extracts in the Erlenneyer flask. Perform a
third extraction in the same manner.
10.5 Assemble a Kuderna-Danish (K-D) concentrator by attaching a 25-mL concentrator tube to a
l000-mL evaporative flask. Other concentration devices or techniques may be used in place of
the K-D if the requirements of Section 8.2 are met.
10.6 Pour the combined extract through a drying column containing about 10 cm of anhydrous sodium
sulfate, and collect the extract in the K-D concentrator. Rinse the Erlenmeyer flask and column
with 20 to 30 mL of methylene chloride to complete the quantitative transfer. Once the flask rinse
has passed through the drying column, rinse the column with 30 to 40 mL of methylene chloride.
10.7 Add one or two clean boiling chips to the evaporative flask and attach a three-ball Snyder column.
Prewet the Snyder column by adding about 1 mL methylene chloride to the top. Place the K-I)
apparatus on a hot water bath, 60 to 65°C, so that the concentrator tube is partially immersed in
the hot water, and the entire lower rounded surface of the flask is bathed with hot vapor. Adjust
the vertical position of the apparatus and the water temperature as required to complete the
concentration in approximately 60 minutes. At the proper rate of distillation, the balls of the
column will actively chatter but the chambers will not flood with condensed solvent. When the
apparent volume of liquid reaches 1 mL, remove the K-I) apparatus and allow it to drain and cool
for at least 10 minutes.
10.8 Increase the temperature of the hot water bath to about 80°C. Momentarily remove the Snyder
column, add 15 mL of methanol and a new boiling chip, and attach a micro-Snyder. Pour about
1 mL of methanol into the top of the micro-Snyder column and concentrate the solvent extract as
before. Elapsed time of concentration should be 5 to 10 minutes. When the apparent volume of
liquid reaches 1 mL, remove the K-D apparatus and allow it to drain and cool for at least 10
minutes.
10
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Method 604.1
10.9 Remove the micro-Snyder column and adjust the volume to 2.5 mL with methanol. Transfer the
liquid to a 5-mL volumetric flask and dilute to the mark with reagent water. Mix thoroughly prior
to analysis. If the extracts will not be analyzed immediately, they should be transferred to PTFE-
sealed screw-cap vials and refrigerated. If the sample extract requires no further cleanup, proceed
with liquid chromatographic analysis. If the sample requires additional cleanup, proceed to
Section 11.
1O.l0Determine the original sample volume by refilling the sample bottle to the mark and transferring
the water to a 1000-mL graduated cylinder. Record the sample volume to the nearest 5 mL.
11. CLEANUP AND SEPARA TION
11 .1 Cleanup procedures may not be necessary for a relatively clean sample matrix. The cleanup
procedure recommended in this method, namely the acid/base extraction described in Section tO,
has been used for the analysis of various clean waters and industrial effluents. If particular
circumstances demand the use of additional cleanup, the analyst must demonstrate that the
recovery of each compound of interest is no less than 85%.
72. LIQuID CHROMATOGRAPHY
12.1 Table 1 summarizes the recommended operating conditions for the liquid chromatograph.
Included in this table are estimated retention times and method detection limits that can be
achieved by this method. An example of the separation achieved by Column 1 is shown in
Figure 1. Examples of the separation achieved by Column 2 are shown in Figure 2. Other
columns, chromatographic conditions, or detectors may be used if the requirements of Section 8.2
are met.
12.2 Calibrate the liquid chromatographic system daily as described in Section 7.
12.3 If an internal standard approach is being used, the analyst must not add the internal standard until
immediately before injection into the instrument. Mix thoroughly.
12.4 Inject 20 to 50 L of the sample extract by completely filling the sample valve loop. Record the
resulting peak sizes in area or peak height units. An automated system that consistently injects
a constant volume of extract may also be used.
12.5 The width of the retention-time window used to make identifications should be based upon
measurements of actual retention time variations of standards over the course of a day. Three
times the standard deviation of a retention time for a compound can be used to calculate a
suggested window size; however, the experience of the analyst should weigh heavily in the
interpretation of chromatograms.
12.6 If the response for the peak exceeds the working range of the system, dilute the extract and
reanalyze.
12.7 If the measurement of the peak response is prevented by the presence of interferences, further
cleanup is required.
11
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Method 604.1
13.
CALCULA 71ONS
13.1
Determine the concentration of individual compounds in the sample.
13.1.1 If the external standard calibration procedure is used, calculate the amount of material
injected from the peak response using the calibration curve or calibration factor in
Section 7.2.2. The concentration in the sample can be calculated as follows:
Equation 2
Concentration, p g/L = ( A)(V )
(V )(V,)
where
A = Amount of material injected, in ng
V. = Volume of extract injected, in *L
= Volume of total extract, in 1 L
V 5 = Volume of water extracted, in mL
13.1.2 If the internal standard calibration procedure was used, calculate the concentration in the
sample using the response factor (RF) determined in Section 7.3.2 as follows:
Equation 3
Concentration, p gIL =
(A )(RF)(V 0 )
where
A Response for parameter to be measured
A = Response for the internal standard
I, Amount of internal standard added to each extract, in g
V 0 Volume of water extracted, in L
13.2 Report results in micrograms per liter without correction for recovery data. When duplicate and
spiked samples are analyzed, report all data obtained with the sample results.
13.3 For samples processed as part of a set where the laboratory spiked sample recovery falls outside
of the control limits in Section 8.3, data for the affected compounds must be labeled as suspect.
14. MEmcOo PERFORMANCE
14.1 The method detection limit (MDL) is defined as the mininum concentration of a substance that can
be measured and reported with 99% confidence that the value is above zero.’ The MDL
concentrations listed in Table I were obtained using reagent water. 1 Similar results were achieved
using representative wastewaters.
14.2 This method has been tested for linearity of recovery from spiked reagent water and has been
demonstrated to be applicable over the concentration range from 10 x MDL to 1000 x MDL.
12
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Method 604.1
14.3 In a single laboratory, Battelle’s Columbus Laboratories, using spiked wastewater samples, the
average recoveries presented in Table 2 were obtained. Seven replicates of each of two different
wastewaters were spiked and analyzed. The standard deviation of the percent recovery is also
included in Table 2.’
13
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Method 604.1
References
1. “Development of Methods for Pesticides in Wastewaters, EPA Contract Report 68-03-2956 (In
preparation).
2. ASTM Annual Book of Standards, Part 31, D3694, “Standard Practice for Preparation of Sample
Containers and for Preservation,” American Society for Testing and Materials, Philadelphia, PA,
p. 679, 1980.
3. “Carcinogens - Working with Carcinogens,” Department of Health, Education, and Welfare,
Public Health Service, Center for Disease Control, National Institute for Occupational Safety and
Health, Publication No. 77-206, August, 1977.
4. “OSHA Safety and Health Standards, General Industry,” (29 CFR 1910), Occupational Safety and
Health Administration, OSHA 2206 (Revised, January 1976).
5. “Safety in Academic Chemistry Laboratories,” American Chemical Society Publication,
Committee on Chemical Safety, 3rd Edition, 1979.
6. “Handbook for Analytical Quality Control in Water and Wastewater Laboratories,”
EPA-60014-79-019, U. S. Environmental Protection Agency, Environmental Monitoring and
Support Laboratory - Cincinnati, Ohio 45268, March 1979.
7. ASTM Annual Book of Standards, Part 31, D3370, “Standard Practice for Sampling Water,”
American Society for Testing and Materials, Philadelphia, PA, p. 76, 1980.
8. Chaser, J. A. et. a!, “Trace Analysis for Wastewaters,” Environmental Science and Technology,
15, 1426 (1981).
14
-------
Method 604.1
Table 1. Chromatographic Conditions and Method Detection Limits
Retention Time (mm) Method Detection
Limit
Parameter Column 1 Column 2 (pg/U
Dichiorophen 4.2 8.2 1.0
Hexachiorophene 9.7 14.4 1.2
Dichiorophen.. Hexachiorophene
Column 1 conditions: Spherisorb-ODS, 5 p , 250 mm long by 4.6 mm; 1 mLlmin flow; 65/35
acetonitrilelwater, 0.05% H3P04. A UV detector was used with this column to determine the MDL.
Column 2 conditions: Lichrosorb RP-2, 5 p. 250 mm long by 4.6 mm; 1 mL/min flow; 50/50
acetonitrile/water, 0.5 acetic acid.
Table 2. Single-Laboratory Accuracy and Precision(a)
Sample Background Spike Mean Standard Number of
Parameter Type (bi pg/U Level Recovery Deviation Replicates
pg/L (%) (%)
Dichlorophen 1 ND (c) 10 58 12.4 7
1 ND 50 107 3.9 7
Hexachiorophene 1 ND 10 82 2.7 7
1 ND 50 102 5.8 7
(a) Column 1 conditions were used.
(b) 1 = POTW secondary effluent
(c) ND = Not detected
15
-------
&bthcd 60L
Figure 1.
HPLC-UV Chromatograrn of 10 ng Each of Hexachiorophefle and
Dichlorophefl (Column 1).
,Dichbrophen
6.0
8.0
Retention Time (minutes)
12.0
14.0
20.0
1 6
-------
Method 604.1
Hexachiorophene
0 2.0 4.0 6.0 8.0 10.0 12.0 14.0 16.0 18.0 20.0
Retention Time (minutes)
52-C 1022A
Figure 2. HPLC-UV Chromatogram of 250 ng Each of Hexachiorophene (Column 2).
17
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Method 608.1
The Determination of
Organochiorine Pesticides in
Municipal and Industrial
Waste water
-------
Method 608.1
The Determination of Organochiorine Pesticides in Municipal and
Industrial Wastewater
SCOPE AND APPLICATION
1 .1 This method covers the determination of certain organochiorine pesticides. The following
parameters can be determined by this method:
Parameter S TORET No. CAS No.
Chlorobenzilate 39460 510-15-6
Chloroneb 2675-77-6
Chloropropylate 5836-40-2
Dibromochloropropane 96-12-8
Etridiazole 2593-15-9
PCNB 82-68-8
Propachior 1918-16-7
1.2 This is a gas chromatographic (GC) method applicable to the determination of the compounds
listed above in industrial and municipal discharges as provided under 40 CFR 136.1. Any
modification of this method beyond those expressly permitted shall be considered a major
modification subject to application and approval of alternative test procedures under 40 CFR 136.4
and 136.5.
1.3 The estimated method detection limit (MDL, defined in Section 125) for each parameter is listed
in Table 1. The MDL for a specific wastewater may differ from those listed, depending upon the
nature of interferences in the sample matrix.
1.4 This method presents an extension in scope of Method 608. Further, the sample extraction and
concentration steps in this method are essentially the same as several others in the 600-series
methods. Thus, a single sample may be extracted to measure the parameters included in the scope
of each of these methods. When cleanup is required, the concentration levels must be high
enough to permit selecting aliquots, as necessary, in order to apply appropriate cleanup
procedures. Under gas chromatography, the analyst is allowed the latitude to select
chromatographic conditions appropriate for the simultaneous measurement of combinations of
these parameters (see Section 12).
1.5 This method is restricted to use by or under the supervision of analysts experienced in the use of
gas chromatography and in the interpretation of gas chromatograms. Each analyst must
demonstrate the ability to generate acceptable results with this method using the procedure
described in Section 8.2.
1.6 When this method is used to analyze unfamiliar samples for any or all of the compounds above,
compound identifications should be supported by at least one additional qualitative technique.
This method describes analytical conditions for a second gas chromatographic column for
chlorobenzilate and chioropropylate that can be used to confirm measurements made with the
21
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Method 608.1
primary column. Section 14 provides gas chromatograph/mass spectrometer (GC/MS) criteria
appropriate for the qualitative confirmation of compound identifications.
2. SUMMARY OF METHOD
2.1 A measured volume of sample, approximately 1 L, is extracted with 15% methylene chloride
using a separatory funnel. The methylene chloride extract is dried and exchanged to hexane
during concentration to a volume of 10 mL or less. Gas chromatographic conditions are described
which permit the separation and measurement of the compounds in the extract by electron capture
(EC) gas chromatography.’
2.2 This method provides an optional Florisil column cleanup procedure to aid in the elimination or
reduction of interferences which may be encountered.
3. INTERFERENCES
3.1 Method interferences may be caused by contaminants in solvents, reagents, glassware, and other
sample-processing apparatus that lead to discrete artifacts or elevated baselines in gas
chromatograms. All reagents and apparatus must be routinely demonstrated to be free from
interferences under the conditions of the analysis by running laboratory reagent blanks as
described in Section 8.5.
3.1.1 Glassware must be scrupulously cleaned. 2 Clean all glassware as soon as possible after
use by’ thoroughly rinsing with the last solvent used in it. Follow by washing with hot
water and detergent and thorough rinsing with tap and reagent water. Drain dry, and
heat in an oven or muffle furnace at 400°C for 15 to 30 minutes. Do not heat volumetric
ware. Thermally stable materials, such as PCBs, may not be eliminated by this
treatment. Thorough rinsing with acetone and pesticide-quality hexane may be substituted
for the heating. After drying and cooling, seal and store glassware in a clean
environment to prevent any accumulation of dust or other contaminants. Store inverted
or capped with aluminum foil.
3.1.2 The use of high-purity reagents and solvents helps to minimize interference problems.
Purification of solvents by distillation in all-glass systems may be required.
3.2 Interferences by phthalate esters can pose a major problem in pesticide analysis when the EC
detector is used. These compounds generally appear in the chromatogram as large late-eluting
peaks, especially in the 15 and 50% fractions from the Florisil column cleanup. Common flexible
plastics contain varying amounts of pbthalates. These phthalates are easily extracted or leached
from such materials during laboratory operations. Cross-contamination of clean glassware occurs
when plastics are handled during extraction steps, especially when solvent-wetted surfaces are
h2ndled. Interferences from phthalates can be minimized by avoiding the use of plastics in the
laboratory. Exhaustive cleanup of reagents and glassware may be required to eliminate
background phthalate contaminafion. 34 The interferences from phthalate esters can be avoided by
using a microcoulometric or electrolytic conductivity detector.
3.3 Matrix interferences may be caused by cont2minants that are coextracted from the sample. The
extent of matrix interferences will vary considerably from source to source, depending upon the
nature and diversity of the industrial complex or municipality sampled. The cleanup procedure
22
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Method 608.1
in Section 11 can be used to overcome many of these interferences, but unique samples may
require additional cleanup approaches to achieve the MDL listed in Table 1.
4. SAFETY
4.1 The toxicity or carcinogenicity of each reagent used in this method has not been precisely defined;
however, each chemical compound must be treated as a potential health hazard. From this
viewpoint, exposure to these chemicals must be reduced to the lowest possible level by whatever
means available. The laboratory is responsible for maintaining a current awareness file of OSfIA
regulations regarding the safe handling of the chemicals specified in this method. A reference file
of material data handling sheets should also be made available to all personnel involved in the
chemical analysis. Additional references to laboratory safety are available and have been
identified for the information of the analyst ! 7
4.2 The following parameters covered by this method have been tentatively classified as known or
suspected human or mammalian carcinogens: chlorobenzilate, dibromochloropropane, and PCNB.
Primary standards of these toxic compounds should be prepared in a hood.
5. APPARA TUS AND MA TERIALS
5.1 Sampling equipment, for discrete or composite sampling.
5.1.1 Grab-sample bottle: Amber borosiicate or flint glass, 1-L or 1-quart volume, fitted with
screw-caps lined with TFE-fluorocarbon. Aluminum foil may be substituted for TFE if
the sample is not corrosive. If amber bottles are not available, protect samples from
light. The container and cap liner must be washed, rinsed with acetone or methylene
chloride, and dried before use to minimize contamination.
5.1.2 Automatic sampler (optional): Must incorporate glass sample containers for the
collection of a minimum of 250 mL. Sample containers must be kept refrigerated at 4°C
and protected from light during compositing. If the sampler uses a peristaltic pump, a
minimum length of compressible silicone rubber tubing may be used. Before use,,
however, the compressible tubing must be thoroughly rinsed with methanol, followed by
repeated rinsings with reagent water to minimize the potential for contamination of the
sample. An integrating flow meter is required to collect flow-proportional composites.
5.2 Glassware. (All specifications are suggested. Catalog numbers are included for illustration only.)
5.2.1 Separatory funnel: 2000-mL, with TFE-fluorocarbon stopcock, ground-glass or TFE
stopper.
5.2.2 Drying column: Chromatographic column 400 mm long by 19 mm ID with coarse-fitted
disc.
5.2.3 Chromatographic column: 400 mm long by 19 mm ID with coarse-fitted disc at bottom
and TFE-fluorocarbon stopcock (Kontes K-420540-0224 or equivalent).
5.2.4 Concentrator tube, Kuderna-Danish: 10-mL, graduated (Kontes K-570050-1025 or
equivalent). Calibration must be checked at the volumes employed in the test. Ground-
glass stopper is used to prevent evaporation of extracts.
5.2.5 Evaporative flask, Kuderna-Danish: 500-mL (Kontes K-570001-0500 or equivalent).
Attach to concentrator tube with springs.
23
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Method 60& 1
5.2.6 Snyder column, Kuderna-Danish: Three-ball macro (Kontes K-503000-0121 or
equivalent).
5.2.7 Vials: Amber glass, 10- to 15-mL capacity with TFE-fluorocarbon-lined screw-cap.
5.3 Boiling chips: Approximately 10140 mesh. Heat at 400°C for 30 minutes or perform a Soxhiet
extraction with methylene chloride.
5.4 Water bath: Heated, with concentric ring cover, capable of temperature control (±2°C). The
bath sliouldbeused inahood.
5.5 Balance: Analytical, capable of accurately weighing to the nearest 0.0001 g.
5.6 Gas chromatograph: Analytical system complete with gas diromatograph suitable for on-column
injection arni all required accessories including syringes, analytical columns, gases, detector, and
strip-chart recorder. A data system is recommended for measuring peak areas.
5.6.1 Column 1: l80cmlongby2mm!Dglass, packedwith 1.5% SP-2250/l.95% SP-2401
on Supelcoport (100/120 mesh) or equivalent. This column was used to develop the
method performance statements in Section 15. Alternative columns may be used in
accordance with the provisions described in Section 12.1.
5.6.2 Column 2: 180 cm long by 2 mm ID glass, packed with Ultrabond 20M (100/120 mesh)
or equivalent.
5.6.3 Detector: Electron capture. This detector has proven effective in the analysis of waste-
waters for the parameters listed in the scope and was used to develop the method
performance statements in Section 15. Alternative detectors, including a mass
spectrometer, may be used in accordance with the provisions described in Section 12.1.
6. REAGENTS
6.1 Reagent water: Reagent water is defined as a water in which an interferent is not observed at the
method detection limit of each parameter of interest.
6.2 Acetone, hexane, isooctane, methylene chloride, methanol: Pesticide-quality or equivalent.
6.3 Ethyl ether: Nanograde, redistilled in glass if necessary. Must be free of peroxides as indicated
by EM Quant test strips, (available from Scientific Products Co., Cat. No. P1126-8, and other
suppliers). Procedures recommended for removal of peroxides are provided with the test strips.
After cleanup, 20 mL ethyl alcohol preservative must be added to each liter of ether.
6.4 Sodium sulfate: ACS, granular, anhydrous. Condition heating in a shallow tray at 400°C for a
minimum of 4 hours to remove phthalates and other interfering organic substances. Alternatively,
heat 16 hours at 450 to 500°C in a shallow tray or perform a Soxhlet extraction with methylene
chloride for 48 hours.
6.5 FlorislI: PR grade (60/100 mesh). Purchase activated at 675°C and store in dark in glass
container with ground-glass stopper or foil-lined screw-cap. Before use, activate each batch at
least 16 hours at 130°C in a foil-covered glass container.
6.6 Stock standard solutions (1.00 pg/giL): Stock standard solutions may be prepared from pure
standard materials or purchased as certified solutions.
6.6.1 Prepare stock standard solutions by accurately weighing approximately 0.0100 g of pure
material. Dissolve the material in pesticide-quality isooctane and dilute to volume in a
24
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Method 608.1
10-mL volumetric flask. Larger volumes may be used at the convenience of the analyst.
If compound purity is certified at 96% or greater, the weight may be used without
correction to calculate the concentration of the stock standard. Commercially prepared
stock standards may be used at any concentration if they are certified by the manufacturer
or by an independent source.
6.6.2 Transfer the stock standard solutions into TFE-fluorocarbon-sealed screw-cap vials.
Store at 4°C and protect from light. Frequently check stock standard solutions for signs
of degradation or evaporation, especially just prior to preparing calibration standards
from them.
6.6.3 Stock standard solutions must be replaced after six months, or sooner if comparison with
check standards indicates a problem.
7 CAL/BRA TION
7.1 Establish gas chromatographic operating parameters equivalent to those indicated in Table 1. The
gas chromatographic system may be calibrated using either the external standard technique
(Section 7.2) or the internal standard technique (Section 7.3).
7.2 External standard calibration procedure:
7.2.1 For each parameter of interest, prepare calibration standards at a minimum of three
concentration levels by adding accurately measured volumes of one or more stock
standards to a volumetric flask and diluting to volume with isooctane. One of the
external standards should be representative of a concentration near, but above, the
method detection limit. The other concentrations should correspond to the range of
concentrations expected in the sample concentrates or should define the working range
of the detector.
7.2.2 Using injections of 1 to 5 L of each calibration standard, tabulate peak height or area
responses against the mass injected. The results can be used to prepare a calibration
curve for each parameter. Alternatively, the ratio of the response to the mass injected,
defined as the calibration factor (CF), may be calculated for each parameter at each
standard concentration. If the relative standard deviation of the calibration factor is less
than 10% over the working range, the average calibration factor can be used in place of
a calibration curve.
7.2.3 The working calibration curve or calibration factor must be verified on each working
shift by the measurement of one or more calibration standards. If the response for any
parameter varies from the predicted response by more than ± 10%, the test must be
repeated using a fresh calibration standard. Alternatively, a new calibration curve or
calibration factor must be prepared for that parameter.
7.3 Internal standard calibration procedure: To use this approach, the analyst must select one or more
internal standards similar in analytical behavior to the compounds of interest. The analyst must
further demonstrate that the measurement of the internal standard is not affected by method or
matrix interferences. Due to these limitations, no internal standard applicable to all samples can
be suggested.
7.3.1 Prepare calibration standards at a minimum of three concentration levels for each
parameter of interest by adding volumes of one or more stock standards to a volumetric
25
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Method 608.1
flask. To each calibration standard, add a known constant amount of one or more
internal standards, and dilute to volume with isooctane. One of the standards should be
representative of a concentration near, but above, the method detection limit. The other
concentrations should correspond to the range of concentrations expected in the sample
concentrates, or should define the working range of the detector.
7.3.2 Using injections of 1 to 5 L of each calibration standard, tabulate the peak height or
area responses against the concentration for each compound and internal standard.
Calculate response factors (RF) for each compound as follows:
Equation 1
RF = (A,)(C,,)
(A ,)(C,)
where
.
A, =
Response for
the parameter to be measured
4, =
Response for
the Internal standard
C , =
Concentration
of the internal standard, In gIL
C,
Concentration
of the parameter to be measured, in ig/L
If the RF value over the working range is constant, less than 10% relative standard
deviation, the RF can be assumed to be invariant and the average RF may be used for
calculations. Alternatively, the results may be used to plot a calibration curve of
response ratios, AJA against RF.
7.3.3 The working calibration curve or RF must be verified on each working shift by the
measurement of one or more calibration standards. If the response for any parameter
varies from the predicted response by more than ± 10%, the test must be repeated using
a fresh calibration standard. Alternatively, a new calibration curve must be prepared for
that compound.
7.4 The cleanup procedure in Section 11 utilizes Florisil chromatography. Florisil from different
batches or sources may vary in adsorptive capacity. To standardize the amount of Florisil which
is used, the use of the lauric acid value is suggested. This procedures determines the adsorption
from hexane solution of lauric acid, in milligram, per gram of Florisil. The amount of Florisil
to be used for each column is calculated by dividing this factor into 110 and multiplying by 20 g.
7.5 Before using any cleanup procedure, the analyst must process a series of calibration standards
through the procedure to validate elutlon patterns and the absence of interference from the
reagents.
8. QUALITY C0Nm0L
8.1 Each laboratory using this method is required to operate a formal quality control program. The
minimum requirements of this program consist of an initial demonstration of laboratory capability
and the analysis of spiked samples as a continuing check on performance. The laboratory is
required to maintain performance records to define the quality of data that is generated.
26
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Method 608.1
8.1 .1 Before performing any analyses, the analyst must demonstrate the ability to generate
acceptable accuracy and precision with this method. This ability is established as
described in Section 8.2.
8.1.2 In recognition of the rapid advances occurring in chromatography, the analyst is
permitted certain options to improve the separations or lower the cost of measurements.
Each time such modifications to the method are made, the analyst is required to repeat
the procedure in Section 8.2.
8.1.3 The laboratory must spike and analyze a minimum of 10% of all samples to monitor
continuing laboratory performance. This procedure is described in Section 8.4.
8.2 To establish the ability to generate acceptable accuracy and precision, the analyst must perform
the following operations.
8.2.1 Select a representative spike concentration for each compound to be measured. Using
stock standards, prepare a quality control check sample concentrate in acetone, 1000
times more concentrated than the selected concentrations.
8.2.2 Using a pipette, add 1.00 mL of the check sample concentrate to each of a minimum of
four 1000-mL aliquots of reagent water. A representative wastewater may be used in
place of the reagent water, but one or more additional aliquots must be analyzed to
determine background levels, and the spike level must exceed twice the background level
for the test to be valid. Analyze the aliquots according to the method beginning in
Section 10.
8.2.3 Calculate the average percent recovery (R), and the standard deviation of the percent
recovery (s), for the results. Wastewater background corrections must be made before
R and s calculations are performed.
8.2.4 Using the appropriate data from Table 2, determine the recovery and single operator
precision expected for the method, and compare these results to the values calculated in
Section 8.2.3. If the data are not comparable, review potential problem areas and repeat
the test.
8.3 The analyst must calculate method performance criteria and define the performance of the
laboratory for each spike concentration and parameter being measured.
8.3.1 Calculate upper and lower control limits for method performance as follows:
Upper Control Limit (UCL) = R + 3s
Lower Control Limit (LCL) = R - 3s
where R and s are calculated as in Section 8.2.3. The UCL and LCL can be used to
construct control charts 9 that are useful in observing trends in performance.
8.3.2 The laboratory must develop and maintain separate accuracy statements of laboratory
performance for wastewater samples. An accuracy statement for the method is defined
as R ± s. The accuracy statement should be developed by the analysis of four aliquots
of wastewater as described in Section 8.2.2, followed by the calculation of R and s.
A.ltenatively, the analyst may use four wastewater data points gathered through the
27
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Method 608.1
requirement for continuing quality control in Section 8.4. The accuracy statements
should be updated regularly. 9
8.4 The laboratory is required to collect in duplicate a portion of their samples to monitor spike
recoveries. The frequency of spiked sample analysis must be at least 10% of all samples or one
spiked sample per month, whichever is greater. One aliquot of the sample must be spiked and
analyzed as described in Section 8.2. If the recovery for a particular parameter does not fall
within the control limits for method performance, the results reported for that parameter in all
samples processed as part of the same set must be qualified as described in Section 13.3. The
laboratory should monitor the frequency of data so qualified to ensure that it remains at or
below 5%.
8.5 Before processing any samples, the analyst must demonstrate through the analysis of a 1-L aliquot
of reagent water that all glassware and reagent interferences are under control. Each time a set
of s2mples is extracted or there is a change in reagents, a laboratory reagent blank must be
processed as a safeguard against laboratory contamination.
8.6 it is recommended that the laboratory adopt additional quality assurance practices for use with this
method. The specific practices that are most productive depend upon the needs of the laboratory
and the nature of the samples. Field duplicates may be analyzed to monitor the precision of the
sampling technique. When doubt exists over the identification of a peak on the chromatogram,
confirmatory techniques such as gas chromatography with a dissimilar column, specific element
detector, or spectrometer must be used. Whenever possible, the laboratory should perform
analysis of quality control materials and participate in relevant performance evaluation studies.
9. SAMPLE COLLECTION, PRESERVATiON, AND HANDLING
9.1 Grab samples must be collected in glass containers. Conventional sampling pract ices 1 ° should be
followed; however, the bottle must not be prerinsed with sample before collection. Composite
samples should be collected in refrigerated glass containers in accordance with the requirements
of the program. Automatic sampling equipment must be as free as possible of plastic and other
potential sources of contamination.
9.2 The samples must be iced or refrigerated at 4°C from the time of collection until extraction.
9.3 All samples must be extracted within 7 days and completely analyzed within 40 days of extraction.
10. SAMPLE EXmAcTION
10.1 Mark the water meniscus on the side of the sample bottle for later determination of sample
volume. Pour the entire sample into a 2-L separatory funnel.
10.2 Add 60 mL methylene chloride to the sample bottle, seal, and shake 30 seconds to rinse the inner
walls. Transfer the solvent to the separatory funnel and extract the sample by shaking the funnel
for 2 minutes with periodic venting to release excess pressure. Allow the organic layer to separate
from the water phase for a minimum of 10 minutes. If the emulsion interface between layers is
more than one-third the volume of the solvent layer, the analyst must employ mechanical
techniques to complete the phase separation. The optimum technique depends upon the sample,
but may include stirring, filtration of the emulsion through glass wool, centrifugation, or other
physical methods. Collect the methylene chloride extract in a 250-mL Erlenmeyer flask.
28
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Method 608.1
10.3 Add a second 60-mL volume of methylene chloride to the sample bottle and repeat the extraction
procedure a second time, combining the extracts in the Erlenmeyer flask. Perform a third
extraction in the same manner.
10.4 Assemble a Kuderna-Danish (K-D) concentrator by attaching a 10-mL concentrator tube to a
500-mL evaporative flask. Other concentration devices or techniques may be used in place of the
K-D if the requirements of Section 8.2 are met.
10.5 Pour the combined extract through a drying column containing about 10 cm of anhydrous sodium
sulfate, and collect the extract in the K-D concentrator. Rinse the Erlenmeyer flask and column
with 20 to 30 mL of methylene chloride to completu the quantitative transfer.
10.6 Add one or two clean boiling chips to the evaporative flask and attach a three-ball Snyder column.
Prewet the Snyder column by adding about 1 mL methylene chloride to the top. Place the K-D
apparatus on a hot water bath, 60 to 65°C, so that the concentrator tube is partially immersed in
the hot water, and the entire lower rounded surface of the flask is bathed with hot vapor. Adjust
the vertical position of the apparatus and the water temperature as required to complete the
concentration in 15 to 20 minutes. At the proper rate of distillation, the balls of the column will
actively chatter but the chambers will not flood with condensed solvent. When the apparent
volume of liquid reaches 1 mL, remove the K-D apparatus and allow it to drain and cool for at
least 10 minutes.
10.7 Increase the temperature of the hot water bath to about 80°C. Momentarily remove the Snyder
column, add 50 mL of hexane and a new boiling chip, and reattach the Snyder column. Pour
about I mL of hexane into the top of the Snyder column and concentrate the solvent extract as
before. Elapsed time of concentration should be 5 to 10 minutes. When the apparent volume of
liquid reaches 1 mL, remove the K-D apparatus and allow it to drain and cool for at least 10
minutes.
10.8 Remove the Snyder column and rinse the flask and its lower joint into the concentrator tube with
1 to 2 mL of hexane and adjust the volume to 10 mL. A 5-mL syringe is recommended for this
operation. Stopper the concentrator tube and store refrigerated if further processing will not be
performed immediately. If the extracts will be stored longer than two days, they should be
transferred to TFE-fluorocarbon-sealed screw-cap vials. If the sample extract requires no further
cleanup, proceed with gas chromatographic analysis. If the sample requires cleanup, proceed to
Section 11.
10.9 Determine the original sample volume by refilling the sample bottle to the mark and transferring
the water to a 1000-mL graduated cylinder. Record the sample volume to the nearest 5 mL.
11. CLEANUP AND SEPARATION
11.1 Cleanup procedures may not be necessary for a relatively clean sample matrix. The cleanup
procedure recommended in this method has been used for the analysis of various industrial and
municipal effluents. If particular circumstances demand the use of an alternative cleanup
procedure, the analyst must determine the elution profile and demonstrate that the recovery of
each compound of interest for the cleanup procedure is no less than 85%.
29
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Method 608.1
11.2 The following Florisil column cleanup procedure has been demonstrated to be applicable to the
four organochiorine pesticides listed in Table 3. It should also be applicable to the cleanup of
extracts for PCNB.
11.2.1 Add a weight of Florisil (nominally 20 g) predetermined by calibration (Section 7.4
and 7.5) to a chromatographic column. Settle the Florisil by tapping the column. Add
anhydrous sodium sulfate to the top of the Florisil to form a layer 1 to 2 cm deep. Add
60 mL of hexane to wet and rinse the sodium sulfate and Florisil. Just prior to exposure
of the sodium sulfate to air, stop the elution of the hexane by closing the stopcock on the
chromatography column. Discard the eluate.
11.2.2 Adjust the sample extract volume to 10 mL with hexane and transfer it from the K-D
concentrator tube to the Florisil column. Rinse the tube twice with 1 to 2 mL hexane,
adding each rinse to the column.
11.2.3 Place a 500-mL K-I) flask and clean concentrator tube under the chromatography
column. Drain the column into the flask until the sodium sulfate layer is nearly exposed.
Elute the column with 200 mL of 6% ethyl ether in hexane (V/V) (Fraction 1) using a
drip rate of about 5 mL/min. Remove the K-D flask and set aside for later
concentration. Elute the column again, using 200 mL of 15% ethyl ether in hexane
(VAT) (Fraction 2), into a second K-D flask. Perform a third elution, using 200 mL of
50% ethyl ether in hexane (V/V) (Fraction 3), into a separate K-D flask. The elution
patterns for four of the pesticides are shown in Table 3.
11.2.4 Concentrate the eluates by standard K-D techniques (Section 10.6), substituting hexane
for the glassware rinses and using the water bath at about 85°C. Adjust final volume to
10 mL with hexane. Analyze by gas chromatography.
12. GAs CHROMATOGRAPHY
12.1 Table I summarizes the recommended operating conditions for the gas chromatograph. Included
in this table are estimated retention times and method detection limits that can be achieved by this
method. Other packed columns, chromatographic conditions, or detectors may be used if the
requirements of Section 8.2 are met. Capillary (open-tubular) columns may also be used if the
relative standard deviations of responses for replicate injections are demonstrated to be less
than 6% and the requirements of Section 8.2 are met.
12.2 Calibrate the system daily as described in Section 7.
12.3 If the internal standard approach is being used, add the internal standard to sample extracts
immediately before injection into the instrument. Mix thoroughly.
12.4 Inject I to 5 pL of the sample extract using the solvent-flush technique. 1 Record the volume
injected to the nearest 0.05 tL, and the resulting peak size in area or peak height units. An
automated system that consistently injects a constant volume of extract may also be used.
12.5 The width of the retention-time window used to make identifications should be based upon
measurements of actual retention-time variations of standards over the course of a day. Three
times the standard deviation of a retention time can be used to calculate a suggested window size
for a compound. However, the experience of the analyst should weigh heavily in the
interpretation of chromatograms.
30
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Method 608.1
12.6 If the response for the peak exceeds the working range of the system, dilute the extract and
reanalyze.
12.7 If the measurement of the peak response is prevented by the presence of interferences, further
cleanup is required.
13. CALCULA TIONS
13.1 Determine the concentration of individual compounds in the sample.
13.1.1 If the external standard calibration procedure is used, calculate the amount of material
injected from the peak response using the calibration curve or calibration factor in Section
7.2.2. The concentration in the sample can be calculated as follows:
Equation 2
( A)(V )
Concentration, zg/L = (V )(V)
where
A = Amount of material injected, in ng
V = Volume of extract infected, In pL
= Volume of total extract, in ‘L
V , = Volume of water extracted, in mL
13.1.2 If the internal standard calibration procedure was used, calculate the concentration in the
sample using the response factor (RF) determined in Section 7.3.2 as follows:
Equation 3
(A,)(l,)
Concentration, g/L (AL,) (RP) (V 0 )
where
A, = Response for parameter to be measured
AL, = Response for the internal standard
I, = Amount of internal standard added to each extract, in g
V 0 = Volume of water extracted, in L
13.2 Report results in micrograms per liter without correction for recovery data. When duplicate and
spiked samples are analyzed, report all data obtained with the sample results.
13.3 For samples processed as part of a set where the laboratory spiked sample recovery falls outside
of the control limits in Section 8.3, data for the affected parameters must be labeled as suspect.
31
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Method 608.1
14. GC/MS CONFIRMA T1ON
14.1 It is recommended that GCIMS techniques be judiciously employed to support qualitative
compound identifications made with this method. The mass spectrometer should be capable of
scanning the mass range from 35 amu to a mass 50 amu above the molecular weight of the
compound. The instrument must be capable of scanning the mass range at a rate to produce at
least 5 scans per peak but not to exceed 7 seconds per scan utilizing a 70 V (nominal) electron
energy in the electron impact ionization mode. A GC to MS interface constructed of all glass or
glass-lined materials is recommended. A computer system should be interfaced to the mass
spectrometer that allows the continuous acquisition and storage on machine-readable media of all
mass spectra obtained throughout the duration of the chromatographic program.
14.2 Gas chromatographic columns and conditions should be selected for optimum separation and
performance. The conditions selected must be compatible with standard GCIMS operating
practices. Chromatographic tailing factors of less than 5.0 must be achieved.’ 2
14.3 At the beginning of each day that confirmatory analyses are to be performed, the GCIMS system
must be checked to see that all decafluorotriphenyl phosphine (DFTPP) performance criteria are
achieved) 3
14.4 To confirm an identification of a compound, the background-corrected mass spectrum of the
compound must be obtained from the sample extract and compared with a mass spectrum from
a stock or calibration standard analyzed under the same chromatographic conditions. It is
recommended thkt at least 25 ng of material be injected into the GCIMS. The criteria below must
be met for qualitative confirmation.
14.4.1 All ions that are present above 10% relative abundance in the mass spectrum of the
standard must be present in the mass spectrum of the sample with agreement to ± 10%.
For example, if the relative abundance of an ion is 30% in the mass spectrum of the
standard, the allowable limits for the relative abundance of that ion in the mass spectrum
for the sample would be 20% to 40%.
14.4.2 The retention time of the compound in the sample must be within 6 seconds of the same
compound in the standard solution.
14.4.3 Compounds that have very similar mass spectra can be explicitly identified by GCIMS
only on the basis of retention time data.
14.5 Where available, chemical ionization mass spectra may be employed to aid in the qualitative
identification process.
14.6 Should these MS procedures fail to provide satisfactory results, additional steps may be taken
before re-analysis. These may include the use of alternative packed or capillary GC columns or
additional cleanup (Section 11).
15. METHOD PERFORMANCE
15.1 The method detection limit (MDL) is defined as the minimum concentration of a substance that
can be measured and reported with 99% confidence that the value is above zero. 14 The MDL
concentrations listed in Table 1 were estimated from the response of an electron capture detector
to each compound. The estimate is based upon the amount of material required to yield a signal
32
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Method 608.1
five times the GC background noise, assuming a 5- 1 L injection from a 1O-mL final extract of a
l-L sample.
15.2 In a single laboratory (West Cost Technical Services, Inc.), using effluents from pesticide
manufacturers and publicly owned treatment works (POTW), the average recoveries presented in
Table 2 were obtained after Florisil cleanup.’ The standard deviations of the percent recoveries
of these measurements are also included in Table 2.
33
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Method 608.1
References
1. “Pesticide Methods Evaluation,” Letter Report #17 for EPA Contract No. 68-03-2697. Available
from U.S. Environmental Protection Agency, Environmental Monitoring and Support Laboratory,
Cincinnati, Ohio 45268.
2. ASTM Annual Book of Standards, Part 31, D3694, “Standard Practice for Preparation of Sample
Containers and for Preservation, “American Society for Testing and Materials, Philadelphia, PA,
p. 679, 1980.
3. Giam, D.S., Chan, H.S. and Nef, G.S., “Sensitive Method for Determination of Phthalate Ester
Plasticizers in Open-Ocean Biota Samples,” Analytical Chemistry, 47, 2225, (1975).
4. Giam, C.S., Chan, H.S., “Control of Blanks in the Analysis of Phthalates in Air and Ocean Biota
Samples,” National Bureau of Standards (U.S.), Special Publication 442, pp. 701-708, 1976.
5. “Carcinogens - Working with Carcinogens,” Department of Health, Education, and Welfare,
Public Health Service, Center for Disease Control, National Institute for Occupational Safety and
Health, Publication No. 77-206, Aug. 1977.
6. “OSHA Safety and Health Standards, General Industry,” (29 CFR 1910), Occupational Safety and
Health Administration, OSHA 2206, (Revised, January 1976).
7. “Safety in Academic Chemistry Laboratories,” American Chemical Society Publication,
Committee on Chemical Safety, 3rd Edition, 1979.
8. ASTM Annual Book of Standards, Part 31, D3086, Appendix X3, “Standardization of Florisil
Column by Weight Adjustment Based on Adsorption of Lauric Acid,” American Society for
Testing and Materials, Philadelphia, PA, p 765, 1980.
9. “Handbook for Analytical Quality Control in Water and Wastewater Laboratories,”
EPA-600/4-79-019, U.S. Environmental Protection Agency, Environmental Monitoring and
Support Laboratory - Cincinnati, Ohio 45268, March 1979.
10. ASTM Annual Book of Standards, Part 31, D3370, “Standard Practice for Sampling Waxer,”
American Society for Testing and Materials, Philadelphia, PA, p. 76, 1980.
11. Burke, l.A., “Gas Chromatography for Pesticide Residue Analysis; Some Practical Aspects,”
Journal of the Association of Official Analytical Chemists, 48, 1037 (1965).
12. McNair, HM. and Boneili, E.J., “Basic Chromatography,” Consolidated Printing, Berkeley,
California, p. 52, 1969.
34
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Method 608.1
References (cont.)
13. Eichelberger, J.W., Harris, L.E., and Budde, W.L. “Reference Compound to Calibrate Ion
Abundance Measurement in Gas Chromatography-Mass Spectrometry,” Analytical Chemistry, 47,
995 (1975).
14. Glaser, J.A., et al., “Trace Analysis for Wastewaters,” Environmental Science & Technology, 15,
1426 (1981).
35
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Method 608.1
Table 1. Chromatographic Conditions and Estimated Method Detection Limits
Column 1 Column 2
__________________________________________ Estimated
Parameter Temperature Retention Time Retention Time MDL
(minutes) (minutes) (pg/Li
Dibromochloropropane 100 3.1 0.04
Etridiazole 140 1.3 0.04
Chioroneb 150 2.0 0.04
Propachlor 150 3.8 1.0
PCNB 160 2.4 0.06
Ch loropropylate 215 3.6 8.4 0.2
Chlorobenzi late 215 3.8 10.7 0.2
Column I conditions: Supelcoport (100/120 mesh) coated with 1.5% SP-2250/1 .95% SP-2401 packed
in a 1.8 m long by 2 mm ID glass column with nitrogen carrier gas at a flow rate of 30 mL/min.
Column temperatures are listed above. An electron capture detector was used with this column to
estimate the MDL.
Column 2 conditions: Ultrabond 20M (100/120 mesh) packed in a 1.8 m long by 2 mm ID glass column
with nitrogen carrier gas at a flow rate of 30 mL/min. Column temperature is 200°C.
36
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Method 608.1
Table 2. Single-Operator Accuracy and Precision
Mean Standard
Sample Background Spike Recovery Deviation Number of
Parameter Type pg/L pg/L (96) (96) Replicates
Chlorobenzilate MW ND 10.5 74 7.2 6
MW ND 52.5 97 3.2 7
Chioroneb MW ND 18.1 92 2.9 7
MW ND 181 93 7.7 7
1W 0.84 6.1 53 38.* 2
1W 110 484 97 18. 2
Chloropropylate MW ND 10.0 78 8.6 6
MW ND 50.0 96 3.3 7
Dibromochloro- MW ND 1.9 83 12.4 7
propane MW ND 24 70 6.5 7
1W ND 1.9 61 — 1
1W ND 24 55 1.2* 2
Etridiazole MW ND 0.50 144 9.9 7
MW ND 9.9 91 1.7 7
PCNB MW ND 1.0 100 11.0 7
MW ND 20.0 91 3.1 7
Propachior 1W 21.3 179 87 3.8 7
MW ND 895 83 3.8 7
ND = Not detected
MW = Municipal wastewater
1W = Industrial wastewater, pesticide manufacturing
* For duplicate analyses, range is listed.
37
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Method 608.1
Table 3. Distribution of Chlorinated Pesticides Into Florisil Column Fractions
Percent Recovery by Fraction
Parameter Fraction 1 Fraction 2 Fraction 3
Chlorobenzilate 0 15 70
Ch loroneb 93
Chioropropylate 0 32 61
Etridiazole 100
Eluant composition by fraction:
Fraction I - 200 mL of 6% ethyl ether in hexane
Fraction 2 - 200 mL of 15% ethyl ether in bexane
Fraction 3 - 200 mL of 50% ethyl ether in hexane
38
-------
Method 608.2
The Determination of Certain
Organochiorine
Pesticides in Municipal and
Industrial Waste water
-------
Method 608.2
The Determination of Certain Organochiorine Pesticides in Municipal
and Industrial Wa ste water
1. SCOPE AND APPIJCA TION
1.1 This method covers the determination of certain organochlorine pesticides in industriaJ and
municipal wastewater. The following parameters may be determined by this method.
Parametea Storet No. CAS No.
Chiorothalonil — 1897-45-6
DCPA 39770 1861-32-1
Dichlorin — 99-30-9
Methoxychlor 39480 72-43-5
Permethrin — 5264553-1
1.2 The estimated detection limit (EDL) for each parameter is listed in Table 1. The EDL was
calculated from the minimum detectable response of the electron capture detector equal to 5 times
the detector background noise assuming a 10.0 -mL final extract volume of a 1-L reagent water
sample and a gas chromatographic (GC) injection volume of 5 1 iL. The EDL for a specific
wastewater may be different depending on the nature of interferences in the sample matrix.
1.3 This is a GC method applicable to the determination of the compounds listed above in municipal
and industrial discharges. When this method is used to analyze unfamiliar samples for any or all
of the compounds listed above, compound identifications should be supported by at least one
additional qualitative technique. Section 13 provides gas chromatograph/mass spectrometer
(GC/MS) conditions appropriate for the qualitative confirmation of compound identifications.
1.4 This method is restricted to use by or under the supervision of analysts experienced in the
operation of gas chromatographs and in the interpretation of chromatograms.
2. SUMMARY OF METHOD
2.1 Organochlorine pesticides are removed from the sample matrix by extraction with methylene
chloride. The extract is dried, exchanged into hexane, and analyzed by gas chromatography.
Column chromatography is used as necessary to eliminate interferences which may be
encountered. Measurement of the pesticides is accomplished with an electron capture detector.
2.2 Confirmatory analysis by gas chromatography/mass spectrometry is recommended (Section 13)
when a new or undefined sample type is being analyzed if the concentration is adequate for such
determination.
41
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Method 608.2
3. INTERFERENCES
3.1 Solvent, reagents, glassware, and other sample-processing hardware may yield discrete artifacts
and/or elevated baselines causing misinterpretation of gas chromatograms. All of these materials
must be demonstrated to be free from interferences under the conditions of the analysis by running
laboratory reagent blanks as described in Section 9.1.
3.1.1 The use of high-purity reagents and solvents helps to minimize interference problems.
Purification of solvents by distillation in all-glass systems may be required.
3.1.2 Glassware must be scrupulously cleaned. 1 Clean all glassware as soon as possible after
use by rinsing with the last solvent used in it. This should be followed by detergent
washing with hot water and rinses with tap water and reagent water. It should then be
drained dry and heated in a muffle furnace at 400°C for 15 to 30 minutes. Solvent rinses
with acetone and pesticide-quality hexane may be substituted for the heating. Volumetric
ware should not be heated in a muffle furnace. After drying and cooling, glassware
should be sealed and stored in a clean environment to prevent any accumulation of dust
or other contaminants. Store the glassware inverted or capped with aluminum foil.
3.2 Interferences coextracted from the samples will vary considerably from source to source,
depending on the diversity of the industrial complex or municipality being sampled. While
general cleanup procedures are provided as part of this method, unique samples may require
additional cleanup approaches to achieve the detection limits listed in Table 1.
4. SAFETY
4.1 The toxicity or carcinogenicity of each reagent used in this method has not been precisely defined;
however, each chemical compound should be treated as a potential health hazard. From this
viewpoint, exposure to these chemicals must be reduced to the lowest possible level by whatever
means available. The laboratory is responsible for maintaining a current awareness file of OSHA
regulations regarding the safe handling of the chemicals specified in this method. A reference file
of material data handling sheets should also be made available to all personnel involved in the
chemical analysis. Additional references to laboratory safety are available and have been
identified for the information of the analyst.
5. APPARATUS AND EQUIPMENT
5.1 Sample containers: Narrow-mouth glass bottles, 1-L or 1-quart volume, equipped with
polytetrafluoroethylene (PTFE)-lined screw-caps. Wide-mouth glass bottles, 1-quart volume,
equipped with PTFE-lined screw-caps may also be used. Prior to use, wash bottles and cap liners
with detergent and rinse with tap and distilled water. Allow the bottles and cap liners to air dry,
then muffle the glass bottles at 400°C for 1 hour. After cooling, rinse the cap liners with hexane,
seal the bottles with aluminum foil, and store in a dust-free environment.
5.1.1
Automatic sampler (optional): Must incorporate glass sample containers for the
collection of a minimum of 250 mL. Sample containers must be kept refrigerated at 4°C
and protected from light during compositing. If the sampler uses a peristaltic pump, a
minimum length of compressible silicone rubber tubing may be used. Before use,
however, the compressible tubing should be thoroughly rinsed with methanol, followed
42
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Method 608.2
by repeated rinsings with distilled water to minimize the potential for contamination of
the sample. An integrating flow meter is required to collect flow-proportional
composites.
5.2 Kuderna-Danjsh (K-D) glassware.
5.2.1 Synder column: Three-ball macro (Kontes K-503000-0121 or equivalent).
5.2.2 Concentrator tube: 10-mL, graduated (Kontes K-570050-1025 or equivalent) with
ground-glass stopper.
5.2.3 Evaporative flask: 500-mL (Kontes K-570001-0500 or equivalent). Attach to
concentrator tube with springs.
5.3 Gas chromatography system.
5.3.1 The gas chromatograph must be equipped with a glass-lined injection port compatible
with the detector to be used. A data system is recommended for measuring peak areas.
5.3.1.1 Column 1: 180 cm long by 2 mm ID, glass, packed with 1.5%
OV-1711.95% OV-210 on Chromosorb W-HP (100/120 mesh) or equivalent.
5.3.1.2 Column 2: 180 cm long by 2 mm ID, glass, packed with 4-% SE-3016-%
SP-2401 on Supelcoport (100/120 mesh) or equivalent. Guidelines for the use
of alternative column packings are provided in Section 10.3.1.
5.3.1.3 Detector: Electron capture. This detector has proven effective in the analysis
of wastewaters for the parameters listed in Section 1.1 and was used to
develop the method performance statements in Section 12. Guidelines for the
use of alternative detectors are provided in Section 10.3.1.
5.4 Chromatographic column: 400 mm long by 19 mm ID Chromaflex, equipped with coarse-fitted
bottom plate and PTFE stopcock. (Kontes K-420540-0224 or equivalent).
Chromatographic column: 300 mm long by 10 mm ID, equipped with coarse-fritted bottom plate
and PTFE stopcock (Kontes K-430540-0213 or equivalent).
5.5 Drying column: Approximately 400 mm long by 20 mm ID borosilicate glass, equipped with
coarse-fitted bottom plate.
5.6 Miscellaneous.
5.6.1 Balance: Analytical, capable of accurately weighing to the nearest 0.0001 g.
5.6.2 Separatory funnel: 2-L, equipped with PTFE stopcock.
5.6.3 Water bath: Heated with concentric ring cover, capable of temperature control (±2°C).
The bath should be used in a hood.
5.6.4 Standard solution storage containers: 15-mL bottles with PTFE-lined screw-caps.
5.6.5 Boiling chips: Approximately 10/40 mesh. Heat to 400°C for 30 minutes, or perform
a Soxhiet extraction overnight with methylene chloride.
43
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Method 6082
6. REAGENTS AND CONSUMABLE MA TEPJALS
6.1 Reagents
6.1.1 Acetone, hexane, ethanol and methylene chloride: Demonstrated to be free of analytes.
6.1.2 Ethyl ether: Nanograde, redistilled in glass if necessary. Must be free of peroxides as
indicated by EM Quant test strips (available from Scientific Products Co., Cat. No.
P1126-8, and other suppliers). Procedures recommended for removal of peroxides are
provided with the test strips. After cleanup, 20 mL ethyl alcohol preservative must be
added to each liter of ether.
6.1.3 Florisil: PR grade (60/100 mesh). Purchase activated at 675°C and store in dark in
glass containers with glass stoppers or foil-lined screw-caps. Before use, activate each
batch overnight at 130°C in foil-covered glass container.
6.1.4 Silica gel: Activate approximately 100 g of silica gel at 200°C for 16 hours in a tared
500-mL Erlenmeyer flask with ground-glass stopper. Allow to cool to room temperature,
and determine the weight of activated silica gel. Deactivate by adding 3% by weight of
distilled water. Restopper the flask, and shake on a wrist-action shaker for at least 1
hour. Allow to equilibrate for 3 or more hours at room temperature.
6.1.5 Reagent water: Reagent water is defined as a water in which an interferent is not
observed at the method detection limit of each parameter of interest.
6.1.6 Sodium hydroxide (NaOH) solution (ION): Dissolve 40 g NaOH in reagent water and
dilute to 100 mL.
6.1.7 Sodium sulfate: Granular, anhydrous. Condition by heating at 400°C for 4 hours in a
shallow tray.
6.1.8 Sulfuric acid (H 2 S0 4 ) solution (1+ l): Add a measured volume of concentrated H 2 S0 4
to an equal volume of reagent water.
6.2 Standard stock solutions (1.00 &gI &L): These solutions may be purchased as certified solutions
or prepared from pure standard materials using the following procedures.
6.2.1 Prepare standard stock solutions by accurately weighing about 0.0100 g of pure material.
Dissolve the material in hexane or other suitable solvent and dilute to volume in a 10-mL
volumetric flask. Larger volumes can be used at the convenience of the analyst. If
compound purity is certified at 96% or greater, the weight can be used without correction
to calculate the concentration of the standard stock.
6.2.2 Store standard stock solutions at 4°C in 15-mL bottles equipped with PTFE-Lined
screw-caps. Standard stock solutions should be checked frequently for signs of
degradation or evaporation, especially just prior to preparing calibration standards from
them.
6.2.3 Standard stock solutions must be replaced after 6 months, or sooner if comparison with
check standards indicates a problem.
44
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Method 608.2
7. SAMPLE COLLECTION, PRESERVATION, AND STORAGE
7.1 Collect all samples in duplicate. Grab samples must be collected in glass containers.
Conventional sampling practices 5 should be followed, except that the bottle must not be prewashed
with sample before collection.
7.2 The samples must be iced or refrigerated at 4°C from the time of collection until extraction.
Chemical preservatives should not be used in the field unless more than 24 hours will elapse
before extraction. If the samples will not be extracted within 48 hours of collection, the sample
should be adjusted to a pH range of 6.0 to 8.0 with sodium hydroxide or sulfuric acid.
7.3 All samples must be extracted within 7 days of collection, and analyzed within 40 days of
extraction. 6
8. CALIBRATION AND STANDARDIZA TZON
8.1 Calibration.
8.1 .1 A set of at least three calibration solutions containing the method analytes is needed. One
calibration solution should contain each analyte at a concentration approaching but greater
than the EDL (Table 1) for that compound; the other two solutions should contain
analytes at concentrations that bracket the range expected in samples. For example, if
the detection limit for a particular analyte is 0.2 pg/L, and a sample expected to contain
approximately 5 g/L is analyzed, standard solutions should be prepared at concentrations
representing 0.3 g/L, 5 g/L, and 10 g/L of the analytes.
8.1 .2 To prepare a calibration solution, add an appropriate volume of a standard stock solution
to a volumetric flask and dilute to volume with hexane.
8.1.3 Starting with the standard of lowest concentration, analyze each calibration standard
according to Section 10.3.2 and tabulate peak height or area response versus the mass of
analyte injected. The results can be used to prepare a calibration curve for each
compound. Alternatively, if the ratio of response to concentration (calibration factor) is
a constant over the working range (< 10% relative standard deviation), linearity through
the origin can be assumed and the average ratio or calibration factor can be used in place
of a calibration curve.
8.1.4 The working calibration curve or calibration factor must be verified on each working day
by the measurement of one or more calibration standards. If the response for any analyte
varies from the predicted response by more than ± 10%, the test must be repeated using
a fresh calibration standard. If the results still do not agree, generate a new calibration
curve.
8.2 Florisil standardization.
8.2.1 Florisil from different batches or sources may vary in absorptive capacity. To
standardize the amount of Florisil which may be used in the cleanup procedure
(Section 10.2.2), use of the lauric acid value 7 is suggested. The referenced procedure
determines the adsorption from hexane solution of lauric acid in milligrams per gram of
Florisil. The amount of Florisil to be used for each column is calculated by dividing this
factor into 110 and multiplying by 20 g.
45
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Method 608.2
9. QUALITY CONTROL
9.1 Monitoring for interferences.
9.1.1 Analyze a laboratory reagent blank each time a set of samples is extracted. A laboratory
reagent blank is a l-L aliquot of reagent water. If the reagent blank contains a reportable
level of any analyte, immediately check the entire analytical system to locate and correct
for possible interferences and repeat the test.
9.2 Assessing accuracy.
9.2.1 After every ten samples, and preferably in the middle of each day, analyze a laboratory
control standard. Calibration standards may not be used for accuracy assessments and
the laboratory control standard may not be used for calibration of the analytical system.
9.2.1.1 Laboratory control standard concentrate: From stock standards prepared as
described in Section 6.3, prepare a laboratory control standard concentrate
that contains each analyte of interest at a concentration of 2 1 ig/mL in acetone
or other suitable solvent.
9.2.1.2 Laboratory control standard: Using a pipette, add 1.00 mL of the laboratory
control standard concentrate to a l-L aliquot of reagent water.
9.2.1.3 Analyze the laboratory control standard as described in Section 10. For each
analyte in the laboratory control standard, calculate the percent recovery (P )
with the equation:
Equation I
l00sI
F =1
= The analytical results from the laboratoly control standard, in ig/L
= The known concentration t( the spike, in tgIL
9.2.2 At least annually, the laboratory should participate in formal performance evaluation
studies, where solutions of HnknOWfl concentrations are analyzed and the performance of
all participants is compared.
9.3 Assessing precision.
9.3.1 Precision assessments for this method are based upon the analysis of field duplicates
(Section 7.1). Analyze both sample bottles for at least 10% of all samples. To the extent
practical, the samples for duplication should contain reportable levels of most of the
analytes.
9.3.2 For each analyte in each duplicate pair, calculate the relative range (R&) with the
on:
46
-------
Method 608.2
Equation 2
= 1OOR
Xi
where
R = The absolute d (ference between the duplicate measurements X 1 and X 2 , in &g/L
x÷x
X = The average concentration found 12 , in gIL
9.3.3 Individual relative range measurements are pooled to determine average relative range
or to develop an expression of relative range as a function of concentration.
10. PROCEDURE
10.1 Sample extraction.
10.1.1 Mark the water meniscus on the side of the sample bottle for later determination of
sample volume. Pour the entire sample into a 2-L separatory funnel. Check the pH of
the sample with wide-range pH paper and adjust to within the range of 5 to 9 with
sodium hydroxide or sulfuric acid.
10.1.2 Add 60 mL of methylene chloride to the sample bottle and shake for 30 seconds to rinse
the walls. Transfer the solvent to the separatory funnel and extract the sample by shaking
the funnel for 2 minutes with periodic venting to release vapor pressure. Allow the
organic layer to separate from the water phase for a minimum of 10 minutes. If the
emulsion interface between layers is more than one-third the volume of the solvent layer,
the analyst must employ mechanical techniques to complete the phase separation. The
optimum technique depends on the sample, but may include stirring, filtration of the
emulsion through glass wool, or centrifugation. Collect the extract in a 250-mL
Erlenmeyer flask.
10.1.3 Add an additional 60-mL volume of methylene chloride to the sample bottle and complete
the extraction procedure a second time, combining the extracts in the Erlenmeyer flask.
10.1.4 Perform a third extraction in the same manner. Pour the combined extract through a
drying column containing about 10 cm of anhydrous sodium sulfate, and collect the
extract in a 500-mL K-D flask equipped with a 10 mL concentrator tube. Rinse the
Erlenmeyer flask and column with 20 to 30 mL of methylene chloride to complete the
quantitative transfer.
10.1.5 Add one or two clean boiling chips to the flask and attach a three-ball Snyder column.
Prewet the Snyder column by adding about 1 mL of methylene chloride to the top. Place
the K-D apparatus on a hot water bath (60 to 65°C) so that the concentrator tube is
partially immersed in the hot water and the entire lower rounded surface of the flask is
bathed in steam. Adjust the vertical position of the apparatus and the water temperature
as required to complete the concentration in 15 to 20 minutes. At the proper rate of
distillation, the balls of the column will actively chatter but the chambers will not flood.
47
-------
Method 608.2
When the apparent volume of liquid reaches about 3 mL, remove the K-I) apparatus and
allow it to drain and cool for at least 10 minutes.
10.1.6 Increase the temperature of the hot water bath to about 80 to 85°C. Momentarily remove
the Snyder column, add 50 mL of hexane and a new boiling chip, and reattach the
Snyder column. Pour about I mL of hexane into the top of the Snyder column, and
concentrate the solvent extract as before. Elapsed time of concentration should be 5 to
10 minutes. When the apparent volume of liquid reaches about 3 mL, remove the K-D
apparatus, and allow it to drain at least 10 minutes while cooling. Remove the Snyder
column, rinsetheflaskandthelowerjointintotheconcentratortubewith 1 to2 mL of
bexane, and adjust the volume to 10 rnL. A 5-mL syringe is recommended for this
operation. Stopper the concentrator tube, and store refrigerated if further processing will
not be performed immediately. If the extracts will be stored longer than 2 days, they
should be transferred to PTFE-sealed screw-cap bottles. If the sample extract requires
no cleanup, proceed with gas chromatographic analysis.
10.1.7 If the sample requires cleanup, the extract obtained must be divided into two fractions.
One of the fractions is eluted through Florisil for the analysis of dicloran and DCPA.
The other fraction is eluted through silica gel for the analysis of chlorothalonil,
methoxychior, and the permethrins.
10.1.8 Determine the original sample volume by refilling the sample bottle to the mark and
transferring the liquid to a 1,000-mL graduated cylinder. Record the sample volume to
the nearest 5 ml..
10.2 Cliesimp and separation.
10.2.1 Cleanup procedures may not be necessary for a relatively clean sample matrix. The
deanup procedures recommended in this method have been used for the analysis of
various dean waters and municipal effluents. The single-operator precision and accuracy
data in Table 2 were gathered using the recommended cleanup procedures. If particular
circumstances desn nd the use of an alternative cleanup procedure, the analyst must
determine the elution profile and demonstrate that the recovery of each compound of
interest is no Less than that recorded in Table 2.
10.2.2 Florisil column cleanup.
10.2.2.1 Add a weighed amount of Florisil, about 21 g, to a chromatographic column.
The exact weight should be determined by calibration. 7 Tap the column to
settle the Florisil. Add a 1 to 2 cm layer of sodium sulfate above the Florisil.
Rinse the Florisil and sodium sulfate by adding 60 mL of hexane to the
column. Just prior to exposure of the sodium sulfate to air, stop the draining
of the hexane by closing the stopcock on the column. Discard the eluate.
10.2.2.2 Quantitatively, add the fraction of extract chosen for the analysis of dichloran
and DCPA to the column. Drain the column into the flask, stopping just
prior to exposure of the sodium sulfate layer.
10.2.2.3 Elute the column with 200 mL of 6% ethyl ether in hexane (Fraction 1) using
a drip rate of about 5 mLIinin. Remove and discard. Perform a second
elution using 200 mL of 15% ethyl ether in hexane (Fraction 2), collecting
the eluant in a 500-mL K-I) flask equipped with a 10-mL concentrator tube.
48
-------
Method 608.2
10.2.2.4 Concentrate the eluate by standard K-D techniques (Section 10.1.5),
substituting hexane for methylene chloride, and using the water bath at about
85°C. Adjust the final volumes to 10 mL with hexane. Analyze by gas
chromatography.
10.2.3 Silica gel column cleanup.
10.2.3.1 Prepare silica gel columns using a 300 mm by 10 mm II) glass column.
Rinse column with hexane. Add approximately 50 mL of hexane to the
empty column. Add 3.5 g of 3% deactivated silica gel (Section 6.1.4). Pack
by rotating slowly to release air bubbles. Top with 1.5 cm of Na 2 SO 4 . Drain
hexane to the top of the Na 2 SO 4 .
10.2.3.2 Add the fraction of extract chosen for the analysis of chiorothalonil,
methoxychior, and the permethrins to the column. Open the stopcock and
allow it to drain to the surface of the sodium sulfate. Elute with the
following solutions:
Fraction 1: 25 mL of hexane
Fraction 2: 25 mL of 6% MeCI 2 in hexane (V/V)
Fraction 3: 25 mL of 50% MeCl 2 in hexane
10.2.3.3 Collect the Fraction 3 in a 500-mL K-D flask equipped with a 10-mL
concentrator tube, and add 50 mL of hexane. Concentrate on an 85°C water
bath to 10.0 inL as described in Section 10.1.5.
10.2.4 The elution profiles obtained in these studies are listed in Tables 3 and 4 for the
convenience of the analyst. The analyst must determine the elution profiles and
demonstrate that the recovery of each compound of interest is no less than that reported
in Table 2 before the analysis of any samples utilizing these cleanup procedures.
10.2.5 Proceed with gas chromatography.
10.3 Gas chromatographic analysis.
10.3.1 Recommended columns and detector for the gas chromatographic system are described
in Section 5.3.1. Table 1 summarizes the recommended operating conditions for the gas
chromatograph. Included in this table are estimated retention times and detection limits
that can be achieved by this method. Examples of the separations achieved by Column 1
are shown in Figures 1 and 2. Other packed columns, chromatographic conditions, or
detectors may be used if data quality comparable to Table 2 are achieved. Capillary
(open-tubular) columns may also be used if the relative standard deviations of responses
for replicate injections are demonstrated to be less than 6% and data quality comparable
to Table 2 are achieved.
10.3.2 Inject 2 to 5 L of the sample extract using the solvent-flush technique. 9 Record the
volume injected to the nearest 0.05 L, the total extract volume, the fraction of total
extract utilized in each cleanup scheme, and the resulting peak size in area or peak height
units.
10.3.3 The width of the retention-time window used to make identifications should be based
upon measurements of actual retention-time variations of standards over the course of the
day. Three times the standard deviation of a retention time for a compound can be used
49
-------
Method 608.2
to calculate a suggested window size; however, the experience of the analyst should
weigh heavily in the interpretation of chromatograms.
10.3.4 If the response for the peak exceeds the working range of the system, dilute the extract
and reanalyze.
10.3.5 If the measurement of the peak response is prevented by the presence of interferences,
further cleanup is required.
11. CALCULATiONS
11.1 Determine the concentration (C) of individual compounds in the sample in micrograms per liter
with the equation:
Equation 3
c= ( A)(V,)(V )
(V 1 ) (V ) (!,)
ere
A - Amowu of material Injected, In ng
Volume of extract injected, In 1 tL
V,= Voiwne of total extract, In ,uL
= Volume of ,wiier extracted, In mL
= Vaiwne of final extract after dea uq,, in pL
V 1 = Volume of extract utilized for cleanup scheme, in pL
11.2 Report the results for the unknown samples in gfL. Round off the results to the nearest 0.1 pg/L
or two significant figures.
12. METHOD PERFORM4NCE
12.1 Estimated detection limits (EDL) and associated chromatographic conditions are listed in Table 1.10
The detection limits were calculated from the minin im detectable response of the EC detector
equal to S times the background noise, assuming a 10.O-mL final extract volume of a 1-L sample
and a GC injection of S pL.
12.2 SingLe-laboratory accuracy and precision studies were conducted by Environmental Science and
Engineering, Inc., 0 using spiked industrial wastewater samples. The results of these studies are
presented in Table 2.
13. GC/MS CONFIRMATION
13.1 It is recommended that GC/MS techniques be judiciously employed to support qualitative
identifications made with this method. The mass spectrometer should be capable of scanning the
mass range from 35 amu to a mass 50 amu above the molecular weight of the compound. The
instrument must be capable of scanning the mass range at a rate to produce at least 5 scans per
peak, but net to exceed 7 scans per peak utilizing a 70 V (nominal) electron energy in the electron
50
-------
Method 608.2
impact ionization mode. A GC to MS interface constructed of all glass or glass-lined materials
is recommended. A computer system should be interfaced to the mass spectrometer that allows
the continuous acquisition and storage on machine-readable media of all mass spectra obtained
throughout the duration of the chromatographic program.
13.2 Gas chromatographic columns and conditions should be selected for optimum separation and
performance. The conditions selected must be compatible with standard GC/MS operating
practices. Chromatographic tailing factors of less than 5.0 must be achieved. The calculation of
tailing factors is illustrated in Method 625.11
13.3 At the beginning of each day that confirmatory analyses are to be performed, the GC/MS system
must be checked to see that all DFTPP performance criteria are achieved. 12
13.4 To confirm an identification of a compound, the background-corrected mass spectrum of the
compound must be obtained from the sample extract and compared with a mass spectrum from
a stock or calibration standard analyzed under the same chromatographic conditions. It is
recommended that at least 25 ng of material be injected into the GC/MS. The criteria below must
be met for qualitative confirmation.
13.4.1 The molecular ion and other ions that are present above 10% relative
abundance in the mass spectrum of the standard must be present in the mass
spectrum of the sample with agreement to ±10%. For example, if the
relative abundance of an ion is 30% in the mass spectrum of the standard, the
allowable limits for the relative abundance of that ion in the mass spectrum
for the sample would be 20 to 40%.
13.4.2 The retention time of the compound in the sample must be within 6 seconds
of the same compound in the standard solution.
13.4.3 Compounds that have similar mass spectra can be explicitly identified by
GC/MS only on the basis of retention time data.
13.5 Where available, chemical ionization mass spectra may be employed to aid in the qualitative
identification process.
13.6 Should these MS procedures fail to provide satisfactory results, additional steps may be taken
before reanalysis. These may include the use of alternative packed or capillary GC columns or
additional cleanup.
51
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Method 60&2
References
1. ASTM Annual Book of Standards, Part 31, D3694, Standard Practice for Preparation of Sample
Containers and for Preservation,’ American Society for Testing and Materials, Philadelphia, PA,
P. 679, 1980.
2. Carcinogens - Working with Carcinogens,’ Department of Health, Education, and Welfare,
Public Health Service, Center for Disease Control, National Institute for Occupational Safety and
Health, Publication No. 77-206, August 1977.
3. OSBA Safety and Health Standards, General Industry’ (29 CFR 1910), Occupational Safety and
Health Administration, OSHA 2206 (Revised, January 1976).
4. ‘Safety in Academic Chemistry Laboratories,’ American Chemical Society Publication,
Committee on Chemical Safety, 3rd Edition, 1979.
5. ASTM Annual Book of Standards, Part 31, D3370, ‘Standard Practice for Sampling Water,’
American Society for Testing and Materials, Philadelphia, PA, P.76 1980.
6. Test procedures for Pesticides in Wastewaters, EPA Contract Report #68-03-2897. Unpublished
report available from U.S. Environmental Protection Agency, Environmental Monitoring and
Support Laboratory, Cincinnati, Ohio.
7. Mills, P.A., ‘Variation of Floricil Activity: Simple Method for Measuring Adsorbent Capacity
and Its Use in Standardizing Florisil Columnc,’ Journal of the Association of Official Analytical
Ozemists, 51, 19 (1968).
8. ‘Handbook for Analytical Quality Control in Water and Wasiewater Laboratories,’
EPA-600/4-79-019, U.S. Environmental Protection Agency, Environmental Monitoring and
Support Laboratory - Cincinnati, Ohio, March 1979.
9. Burke, J.A., ‘Gas Chromatography fbi Pesticide Residue Analysis; Some Practical Aspects,’
Journal of the Association of Official Analytical Ojemists, 48, 1037(1965).
10. ‘Evaluation of Ten Pesticide Methods,’ Contract Report #68-03-1760, Task No. 11, U.S.
Environmental Protection Agency, Environmental Monitoring and SupportLaboratory, Cincinnati,
Ohio.
11. ‘MethOds for Organic Chemical Analysis of Municipal and Industrial Wastewater,’
EPA-60014-82-057. U.S. Environmental Protection Agency, Environmental Monitoring and
Support Laboratory, Cinchm2ti, Ohio.
12. Eichdberger, J.W., Harris, L.E., and Budde, W.L., Anal. Oem., 46, 1912 (1975).
52
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Method 608.2
Table 1. Gas Chromatography of Organochiorine Pesticides
Retention Time (Mm.) Estimated
Detection
Parameter
Column 1* Column 2* * Limit
Chlorothalonil 3.44) 4.69 0.001
DCPA 4.19 5.44 0.003
Dicloran 2.23 2.62 0.002
Methoxychlor 22.35 10.85 0.04
cis Permethrin*** 18.52 16.04 0.2
trans Permethrin*** 20.02 17.53 0.2
* Column 1: 180 cm long by 2 mm ID, glass, packed with 1.5% OV-17/1.95% OV 210 on
Chromosorb W-HP (1001120 mesh) or equivalent; 5% methanel95% Argon carrier gas at
30 mLlmin flow rate. Column temperature is 200°C. Detector: electron capture.
** Column 2: 180 cm long by 2 mm ID, glass, packed with 4% SE-30/6% SP-2401 on Supelcoport
(100/120 mesh) or equivalent; 5% methane/95% Argon carrier gas at 60 mL/min flow rate.
Column temperature is 200°C. Detector: electron capture.
Column temperature is 220°C.
53
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Method 608.2
Table 2. Single-Laboratory Accuracy and Precision
Spike Average Standard
Metric Range Number of Percent Deviation
Parameter Type* (pg L) ReplicateS Recovery (%)
Chlorothalonil 1 37.8 7 84.1 16.4
2 2,300 7 94.9 22.5
DCPA 1 16 7 77.6 25.7
2 10,540 7 89.5 11.0
Dicloran 1 37.5 7 98.6 8.4
2 21,200 7 90.8 20.3
Methoxychlor 1 24.5 7 102.4 12.4
2 2,600 7 102.2 10.2
cis-Permethrin 1 6.3 7 99.5 18.8
2 317 7 77.5 10.6
trans-Permethrin 1 5.7 7 78.8 16.1
2 297 7 88.9 19.6
* 1 = Low-level industrial effluent
2 = High-level industrial effluent
Table 3. Elutlon Profiles for Florisil Cleanup
Percent Recover By Fraction
Parameter — 1 2 3
DCPA 0 99.3 0
Dicloran 0 96.3 0
* J t ing lvent inposition f each fraction given in Section 10.2.2.3.
54
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Method 608.2
Table 4. Elution Profiles for Silica Gel* Cleanup
Percent Recovery By Fraction * *
Pa,ameter 1 2 3
Chiorothalonil 0 0 93.8
Methoxychior 0 0 93.8
cis-Permethrin 0 0 107.2
trans-Permethrin 0 0 92.5
* 3% deactivated
** Eluting solvent composition for each fraction given in Sections 10.2.3.2 and 10.2.3.3.
55
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/Odoran
/ ChIorotha1on
DCPA
Methoxychior
‘: j i;;IIII11l11
0 2.0 4.0 6.0 8.0 10.0 12.0 14.0 16.0 18.0 20.0 22.0
Retention Thne (minutes)
A5200214
Figure 1. Gas Chromatogram of Chiorothalonil, DCPA, Dicloran,
and Methoxychior in a Wastewater Extract (Column 1).
56
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Method 608.2
Figure 2. Gas Chromatogram of Permethrin Sample (Column 1).
trans-Permethrin
cis-Permethrin /
0 2.0 4.0 6.0 8.0 10.0 12.0
Retention Time (minutes)
14.0 16.0 18.0
20.0
*52.002.13
57
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Method 614
The Determination of
Orgénophosphorus Pesticides in
Municpal and Industrial
Waste water
-------
Method 614
The Determination of Organophosphorus Pesticides in Municipal and
Industrial Wastewater
SCOPE AND APPUCA TION
1.1 This method covers the determination of certain organophosphorus pesticides. The following
parameters can be determined by this method:
Parameter STORET No. CAS No.
Azinphos methyl 39580 86-50-0
Demeton 39560 806548-3
Diazinon 39570 333-41-5
Disulfoton 39010 298-04-4
Ethion 563 -12-2
Malathion 39530 121-75-5
Parathion ethyl 39540 56-38-2
Parathion methyl 39600 298-00-0
1.2 This is a gas chromatographic (GC) method applicable to the determination of the compounds
listed above in industrial and municipal discharges as provided under 40 CFR 136.1. Any
modification of this method beyond those expressly permitted shall be considered a major
modification subject to application and approval of alternative test procedures under 40 CFR 136.4
and 136.5.
1.3 The method detection limit (MDL, defined in Section 15) for several parameters are listed in
Table 1. The MDL for a specific wastewater may differ from those listed, depending upon the
nature of interferences in the sample matrix.
1.4 The sample extraction and concentration steps in this method are essentially the same as in
Method 617. Thus, a single sample may be extracted to measure the parameters included in the
scope of both of these methods. When cleanup is required, the concentration levels must be high
enough to permit selecting aliquots, as necessary, in order to apply appropriate cleanup
procedures. Under gas chromatography, the analyst is allowed the latitude to select
chromatographic conditions appropriate for the simultaneous measurement of combinations of
these parameters (see Section 12).
1.5 This method is restricted to use by or under the supervision of analysts experienced in the use of
gas chromatography and in the interpretation of gas chromatograms. Each analyst must
demonstrate the ability to generate acceptable results with this method using the procedure
described in Section 8.2.
1.6 When this method is used to analyze unfamiliar samples for any or all of the compounds above,
compound identifications should be supported by at least one additional qualitative technique.
This method describes analytical conditions for a second gas chromatographic column that can be
used to confirm measurements made with the primary column. Section 14 provides gas
61
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Method 614
chromatograph/mass spectrometer (GC/MS) criteria appropriate for the qualitative confirmation
of compound identifications.
2. SUMMARY OF METHOD
2.1 A measured volume of sample, approximately 1 L, is extracted with 15% methylene chloride in
hexane using a separatory funnel. The extract is dried and concentrated to a volume of 10 mL
or less. Gas chromatographic conditions are described which permit the separation and
measurement of the compounds in the extract by flame photometric or thermionic bead gas
chromatography.
2.2 Method 614 represents an editorial revision of a previously promulgated U.S. EPA method for
organophosphorus pesticides.’ While complete method validation data is not presented herein, the
method has been in widespread use since its promulgation, and represents the state of the art for
the analysis of such materials.
2.3 This method provides selected cleanup procedures to aid in the elimination of interferences which
may be encountered.
3. INTERFERENCES
3.1 Method interferences may be caused by contaminants in solvents, reagents, glassware, and other
sample-processing apparatus that lead to discrete artifacts or elevated baselines in gas
chroinatograms. AU reagents and apparatus must be routinely demonstrated to be free from
interferences under the conditions of the analysis by running laboratory reagent blanks as
described in Section 8.5.
3.1.1
Glassware must be scrupulously cleaned. 2 Clean all glassware as soon as possible after
use by thoroughly rinsing with the last solvent used in it. Follow by washing with hot
water and detergent and thorough rinsing with tap and reagent water. Drain dry, and heat
in an oven or muffle furnace at 400°C for 15 to 30 minutes. Do not heat volumetric
ware. Thermally stable materials, such as PCBs, may not be eliminated by this
treatment. Thorough rinsing with acetone and pesticide-quality hexane may be substituted
for the beating. After drying and cooling, seal and store glassware in a clean
environment to prevent any accumulation of dust or other contaminants. Store inverted
or capped with aluminum foil.
3.1.2 The use of high-purity reagents and solvents helps to minimize interference problems.
Purification of solvents by distillation in all-glass systems may be required.
3.2 Matrix interferences may be caused by contaminants that are coextracted from the sample. The
extent of matrix interferences will vary considerably from source to source, depending upon the
nature and diversity of the industrial complex or municipality sampled. The cleanup procedure
in Section 11 can be used to overcome many of these interferences, but unique samples may
require additional cleanup approaches to achieve the MDL listed in Table I.
4. SAFETY
4.1 The toxicity or carcinogenicity of each reagent used in this method has not been precisely defined;
however, each chemical compound must be treated as a potential health hazard. From this
62
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Method 614
viewpoint, exposure to these chemicals must be reduced to the lowest possible level by whatever
means available. The laboratory is responsible for maintaining a current awareness file of OSHA
regulations regarding the safe handling of the chemicals specified in this method. A reference file
of material data handling sheets should also be made available to all personnel involved in the
chemical analysis. Additional references to laboratory safety are available and have been
identified for the information of the analyst.
5. APPARA TUS AND MA TERIALS
5.1 Sampling equipment, for discrete or composite sampling.
5.1.1 Grab-sample bottle: Amber borosilicate or flint glass, 1-L or 1-quart volume, fitted with
screw-caps lined with TFE-fluorocarbon. Aluminum foil may be substituted for TFE if
the sample is not corrosive. If amber bottles are not available, protect samples from
light. The container and cap liner must be washed, rinsed with acetone or methylene
chloride, and dried before use to minimize contamination.
5.1.2 Automatic sampler (optional): Must incorporate glass sample containers for the
collection of a minimum of 250 mL. Sample containers must be kept refrigerated at 4°C
and protected from light during compositing. If the sampler uses a peristaltic pump, a
minimum length of compressible silicone rubber tubing may be used. Before use,
however, the compressible tubing must be thoroughly rinsed with methanol, followed by
repeated rinsings with reagent water to minimize the potential for contamination of the
sample. An integrating flow meter is required to collect flow-proportional composites.
5.2 Glassware (All specifications are suggested. Catalog numbers are included for illustration only.)
5.2.1 Separatory funnel: 125-mL, l000-mL, and 2000-mL, with TFE-fluorocarbon stopcock,
ground-glass or TFE stopper.
5.2.2 Drying column: Chromatographic column 400 mm long by 19 mm ID with coarse-fitted
disc.
5.2.3 Chromatographic column: 400 mm long by 19 mm ID with coarse-fitted disc at bottom
and TFE-fluorocarbon stopcock (Kontes K-420540-0224 or equivalent).
5.2.4 Concentrator tube, Kuderna-Danish: 10-mL, graduated (Kontes K-570050-1025 or
equivalent). Calibration must be checked at the volumes employed in the test. Ground-
glass stopper is used to prevent evaporation of extracts.
5.2.5 Evaporative flask, Kuderna-Danish: 500-mL (Kontes K-570001-0500 or equivalent).
Attach to concentrator tube with springs.
5.2.6 Snyder column, Kuderna-Danish: Three-ball macro (Kontes K-503000-0121 or
equivalent).
5.2.7 Snyder column, Kuderna-Danish: Two-ball micro (Kontes K-569001-0219 or
equivalent).
5.2.8 Pipette, disposable: 140 mm long by 5 mm ID.
5.2.9 Vials: Amber glass, 10- to 15-mL capacity with TFE-fluorocarbon-Iined screw-cap.
5.3 Boiling chips: Approximately 10/40 mesh. Heat at 400°C for 30 minutes or perform a Soxhlet
extraction with methylene chloride.
63
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Method 614
5.4 Water bath: Heated, with concentric ring cover, capable of temperature control (±2°C). The
bath should be used in a hood.
5.5 Balance: Analytical, capable of accurately weighing to the nearest 0.0001 g.
5.6 Gas chromatograph: Analytical system complete with gas chromatograph suitable for on-column
injection and all required accessories including syringes, analytical columns, gases, detector, and
strip-chart recorder. A data system is recommended for measuring peak areas.
5.6.1 Column 1: 180 cm long by 4 mm II) glass, packed with 3% OV-1 on Gas Chrom Q
(100/120 mesh) or equivalent. This column was used to develop the method performance
statements in Section 15. Alternative columns may be used in accordance with the
provisions described in Section 12.1.
5.6.2 Column 2: 180 cm long by 4 mm ID glass, packed with 1.5% OV-17/1.95% QF-1 on
Gas Chrom Q (100/120 mesh) or equivalent.
5.6.3 Detector: Phosphorus-specific; flame photometric (FPD) (526 nm filter) or thermionic
bead detector in the nitrogen mode. These detectors have proven effective in the analysis
of wastewaters for the parameters listed in the scope. The FPD was used to develop the
method performance statements in Section 15. Alternative detectors, including a mass
spectrometer, may be used in accordance with the provisions described in Section 12.1.
6. REAGENTS
6.1 Reagent water: Reagent water is defined as a water in which an interferent is not observed at the
method detection limit of each parameter of interest.
6.2 Acetone, hexane, isooctane, methylene chloride: Pesticide-quality or equivalent.
6.3 Ethyl ether: Nanograde, redistilled in glass if necessary. Must be free of peroxides as indicated
by EM Quant test strips (available from Scientific Products Co., Cat. No. P1126-8, and other
suppliers). Procedures recommended for removal of peroxides are provided with the test strips.
After cleanup, 20 mL ethyl alcohol preservative must be added to each liter of ether.
6.4 Acetonitrile, hexane-saturated: Mix pesticide-quality acetonitrile with an excess of hexane until
equilibrium is established.
6.5 Sodium sulfate: ACS, granular, anhydrous. Condition by heating in a shallow tray at 400°C for
a minimum of 4 hours to remove phthalates and other interfering organic substances.
Alternatively, beat 16 hours at 450 to 500°C in a shallow tray or perfonn a Soxhlet extraction
with methylene chloride for 48 hours.
6.6 Sodium chloride solution, saturated: Prepare saturated solution of NaCI in reagent water and
extract with bexane to remove impurities.
6.7 Alumina: Woelm, neutral; deactivate by pipetting 1 mL of distilled water into a 125-mL ground-
glass stoppered Erlenmeyer flask. Rotate flask to distribute water over surface of glass.
Immediately add 19.0 g fresh alumina through small powder funnel. Shake flask containing
mixture for 2 hours on a mechanical shaker.
6.8 Florisil: PR grade (60/100 mesh). Purchase activated at 675°C and store in dark in glass
container with ground-glass stopper or foil-lined screw-cap. Before use, activate each batch at
least 16 hours at 130°C in a foil-covered glass container.
64
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Method 614
6.9 Stock standard solutions (1.00 pgI L): Stock standard solutions may be prepared from pure
standard materials or purchased as certified solutions.
6.9.1
Prepare stock standard solutions by accurately weighing approximately 0.0100 g of pure
material. Dissolve the material in pesticide-quality isooctane or acetone and dilute to
volume in a 10-mL volumetric flask. Larger volumes may be used at the convenience
of the analyst. If compound purity is certified at 96% or greater, the weight may be used
without correction to calculate the concentration of the stock standard. Commercially
prepared stock standards may be used at any concentration if they are certified by the
manufacturer or by an independent source.
6.9.2 Transfer the stock standard solutions into TFE-fluorocarbon-sealed screw-cap vials.
Store at 4°C and protect from light. Frequently check stock standard solutions for signs
of degradation or evaporation, especially just prior to preparing calibration standards
from them.
6.9.3 Stock standard solutions must be replaced after 6 months, or sooner if comparison with
check standards indicates a problem.
7. CALIBRA TION
7.1 Establish gas chromatographic operating parameters equivalent to those indicated in Table 1. The
gas chromatographic system may be calibrated using either the external standard technique
(Section 7.2) or the internal standard technique (Section 7.3).
7.2 External standard calibration procedure.
7.2.1 For each parameter of interest, prepare calibration standards at a minimum of three
concentration levels by adding accurately measured volumes of one or more stock
standards to a volumetric flask and diluting to volume with isooctane or other suitable
solvent. One of the external standards should be representative of a concentration near,
but above, the method detection limit. The other concentrations should correspond to the
range of concentrations expected in the sample concentrates or should define the working
range of the detector.
7.2.2 Using injections of 1 to 5 L of each calibration standard, tabulate peak height or area
responses against the mass injected. The results can be used to prepare a calibration
curve for each parameter. Alternatively, the ratio of the response to the mass injected,
defined as the calibration factor (CF), may be calculated for each parameter at each
standard concentration. If the relative standard deviation of the calibration factor is less
than 10% over the working range, the average calibration factor can be used in place of
a calibration curve.
7.2.3 The working calibration curve or calibration factor must be verified on each working
shift by the measurement of one or more calibration standards. If the response for any
parameter varies from the predicted response by more than ± 10%, the test must be
repeated using a fresh calibration standard. Alternatively, a new calibration curve or
calibration factor must be prepared for that parameter.
7.3 Internal standard calibration procedure: To use this approach, the analyst must select one or more
internal standards similar in analytical behavior to the compounds of interest. The analyst must
65
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Method 614
further demonstrate that the measurement of the internal standard is not affected by method or
matrix interferences. Due to these limitations, no internal standard applicable to all samples can
be suggested.
7.3.1 Prepare calibration standards at a minimum of three concentration levels for each
parameter of interest by adding volumes of one or more stock standards to a volumetric
flask. To each calibration standard, add a known constant amount of one or more
internal standards, and dilute to volume with isooctane or other suitable solvent. One of
the standards should be representative of a concentration near, but above, the method
detection limit. The other concentrations should correspond to the range of
concentrations expected in the sample concentrates, or should define the working range
of the detector.
7.3.2 Using injections of I to 5 zL of each calibration standard, tabulate the peak height or
area responses against the concentration for each compound and internal standard.
Calculate response factors (RF) for each compound as follows:
Equation 1
RF (A,)(C,)
(A )(C,)
.s*ere
.
A, =
Response for the parameter to be measure
4, =
Response for the internal standani
Cb =
Concentration of the internal standard, in g/L
C, =
Concentration of the parameter to be measured, in g!L
If the RF value over the working range is constant, less than 10% relative standard
deviation, the RF can be assumed to be invariant and the average RF may be used for
calculations. Alternatively, the results may be used to plot a calibration curve of
response ratios, AJA against RF.
7.3.3 The working calibration curve or RF must be verified on each working shift by the
measurement of one or more calibration standards. If the response for any parameter
varies from the predicted response by more than ±10%, the test must be repeated using
a fresh calibration standard. Alternatively, a new calibration curve must be prepared for
that compound.
7.4 The cleanup procedure in Section 11 utilizes Florisil chromatography. Florisil from different
batches or sources may vary in adsorptive capacity. To standardize the amount of Florisil which
is used, the use of the lauric acid value is suggested. This procedure 6 determines the adsorption
from hexane solution of lauric acid, in milligrams, per gram of Florisil. The amount of Florisil
to be used for each column is calculated by dividing this factor into 110 and multiplying by 20 g.
7.5 Before using any cleanup procedure, the analyst must process a series of calibration standards
through the procedure to validate elution patterns and the absence of interference from the
reagents.
66
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Method 614
8. QUALITY CONTROL
8.1 Each laboratory using this method is required to operate a formal quality control program. The
minimum requirements of this program consist of an initial demonstration of laboratory capability
and the analysis of spiked samples as a continuing check on performance. The laboratory is
required to maintain performance records to define the quality of data that is generated.
8.1.1 Before performing any analyses, the analyst must demonstrate the ability to generate
acceptable accuracy and precision with this method. This ability is established as
described in Section 8.2.
8.1.2 In recognition of the rapid advances occurring in chromatography, the analyst is
permitted certain options to improve the separations or lower the cost of measurements.
Each time such modifications to the method are made, the analyst is required to repeat
the procedure in Section 8.2.
8.1.3 The laboratory must spike and analyze a minimum of 10% of all samples to monitor
continuing laboratory performance. This procedure is described in Section 8.4.
8.2 To establish the ability to generate acceptable accuracy and precision, the analyst must perform
the following operations.
8.2.1
Select a representative spike “ for each compound to be measured. Using
stock standards, prepare a quality control check sample concentrate in acetone, 1000
times more concentrated -than the selected concentrations.
8.2.2 Using a pipette, add 1.00 mL of the check sample concentrate to each of a minimum of
Ibur l000-mL aliquots of reagent water. A representative wastewater may be used in
place of the reagent water, but one or more additional aliquots must be analyzed to
determine background levels, and the spike level must exceed twice the background level
for the test to be valid. Analyze the aliquots according to the method beginning in
Section 10.
8.2.3 Calculate the average percent recovery (R), and the standard deviation of the percent
recovery (s), for the results. Wastewater background corrections must be made before
R and calculations are performed.
8.2.4 Table 2 provides single-operator recovery and precision for diazinon, parathion methyl,
and parathion ethyl. Similar results should be expected from reagent water for all
organophosphorus compounds listed in this method. Compare these results to the values
calculated in Section 8.2.3. If the data are not comparable, review potential problem
areas and repeat the test.
8.3 The analyst must calculate method performance criteria and define the performance of the
laboratory for each spike concentration and parameter being measured.
8.3.1 Calculate upper and lower control limits for method performance as follows:
JU’... 1ILL QLLJLI
Upper Control Limit (UCL) = R .+ 3s
Lower Control Limit (LCL) = R -3s
where R and S are calculated as in Section 8.2.3. The UCL and LCL can be used to
construct control charts 7 that are useful in observing trends in performance.
67
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Method 614
8.3.2 The laboratory must develop and maintain separate accuracy statements of laboratory
performance for wastewater samples. An accuracy statement for the method is defined
as R ± s. The accuracy statement should be developed by the analysis of four aliquots
of wastewater as described in Section 8.2.2, followed by the calculation of R and s.
Alternatively, the analyst may use four wastewater data points gathered through the
requirement for continuing quality control in Section 8.4. The accuracy statements
should be updated regularly. 7
8.4 The laboratory is required to collect in duplicate a portion of their samples to monitor spike
recoveries. The frequency of spiked sample analysis must be at least 10% of all samples or one
spiked sample per month, whichever is greater. One aliquot of the sample must be spiked and
analyzed as described in Section 8.2. If the recovery for a particular parameter does not fall
within the control limits for method performance, the results reported for that parameter in all
samples processed as part of the same set must be qualified as described in Section 13.3. The
laboratory should monitor the frequency of data so qualified to ensure that it remains at or
below 5%.
8.5 Before processing any samples, the analyst must demonstrate through the analysis of a l-L aliquot
of reagent water that all glassware and reagent interferences are under control. Each time a set
of samples is extracted or there is a ch2nge in reagents, a laboratory reagent blank must be
processed as a safeguard against laboratory contamination.
8.6 It is recom* ended that the laboratory adopt additional quality assurance practices for use with this
method. The specific practices that are most productive depend upon the needs of the laboratory
and the nature of the samples. Field duplicates may be analyzed to monitor the precision of the
sampling technique. When doubt exists over the identification of a peak on the chromatogram,
confirmatory techniques such as gas chromatography with a dissimilar column, specific element
detector, or mass spectrometer must be used. Whenever possible, the laboratory should perform
analysis of quality control materials and participate in relevant performance evaluation studies.
9. SAMPLE COLLECTiON, PRESERVATiON, AND HANDLING
9.1 Grab samples must be collected in glass containers. Conventional sampling practices’ should be
followed; however, the bottle must not be prerinsed with sample before collection. Composite
samples should be collected in refrigerated glass containers in accordance with the requirements
of the program. Automatic sampling equipment must be as free as possible of plastic and other
potential sources of contamination.
9.2 The samples must be iced or refrigerated at 4°C from the time of collection until extraction.
9.3 All samples must be extracted Within 7 days and completely analyzed within 40 days of extraction.
10. SAMPLE ExTRACTION
10.1 Mark the water meniscus on the side of the sample bottle for later determination of sample
volume. Pour the entire sample into a 2-L separatory funnel.
10.2 Add 60 niL 15% methylene chloride in hexane (V:V) to the sample bottle, seal, and shake
30 seconds to rinse the inner walls. Transfer the solvent to the separatory funnel and extract the
sample by shaking the funnel for 2 minutes with periodic venting to release excess pressure.
68
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Method 614
Allow the organic layer to separate from the water phase for a minimum of 10 minutes. If the
emulsion interface between layers is more than one third the volume of the solvent layer, the
analyst must employ mechanical techniques to complete the phase separation. The optimum
technique depends upon the sample, but may include stirring, filtration of the emulsion through
glass wool, centrifligation, or other physical methods. Drain the aqueous phase into a l000-mL
Erlenmeyer flask and collect the extract in a 250-mL Erlenmeyer flask. Return the aqueous phase
to the separatory funnel.
10.3 Add a second 60-mL volume of 15% methylene chloride in hexane to the sample bottle and repeat
the extraction procedure a second time, combining the extracts in the 250-mL Erlenmeyer flask.
Perform a third extraction in the same manner.
10.4 Assemble a Kuderna-Danish (K-D) concentrator by attaching a 10-mL concentrator tube to a
500-mL evaporative flask. Other concentration devices or techniques may be used in place of the
K-D if the requirements of Section 8.2 are met.
10.5 Pour the combined extract through a drying column containing about 10 cm of anhydrous sodium
sulfate, and collect the extract in the K-D concentrator. Rinse the Erlenmeyer flask and column
with 20 to 30 mL of hexane to complete the quantitative transfer.
10.6 Add one or two clean boiling chips to the evaporative flask and attach a three-ball Snyder column.
Prewet the Snyder column by adding about 1 mL methylene chloride to the top. Place the K-D
apparatus on a hot water bath, 80 to 85°C, so that the concentrator tube is partially immersed in
the hot water, and the entire lower rounded surface of the flask is bathed with hot vapor. Adjust
the vertical position of the apparatus and the water temperature as required to complete the
concentration in 15 to 20 minutes. At the proper rate of distillation, the balls of the column will
actively chatter but the chambers will not flood with condensed solvent. When the apparent
volume of liquid reaches 1 mL, remove the K-D apparatus and allow it to drain and cool for at
least 10 minutes.
10.7 Remove the Snyder column and rinse the flask and its lower joint into the concentrator tube with
1 to 2 mL of hexane and adjust the volume to 10 mL. A 5-mL syringe is recommended for this
operation. Stopper the concentrator tube and store refrigerated if further processing will not be
performed immediately. If the extracts will be stored longer than two days, they should be
transferred to PTFE-sealed screw-cap bottles. If the sample extract requires no further cleanup,
proceed with gas chromatographic analysis. If the sample requires cleanup, proceed to
Section 11.
10.8 Determine the original sample volume by refilling the sample bottle to the mark and transferring
the water to a 1000-mL graduated cylinder. Record the sample volume to the nearest 5 mL.
11. CLEANUP AND SEPARA TION
11.1 Cleanup procedures may not be necessary for a relatively clean sample matrix. The cleanup
procedure recommended in this method has been used for the analysis of various industrial and
municipal effluents. If particular circumstances demand the use of an alternative cleanup
procedure, the analyst must determine the elution profile and demonstrate that the recovery of
each compound of interest for the cleanup procedure is no less than 85%.
69
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Method 614
11.2 Acetonitrile partition: The following acetonitrile partitioning procedure may be used to isolate fats
and oils from the sample extracts. The applicability of this procedure to organophosphorus
pesticides is indicated in Table 3.
11.2.1 Quantitatively transfer the previously concentrated extract to a 125-mL separatory funnel
with enough hexane to bring the final volume to 15 mL. Extract the sample four times
by shaking vigorously for 1 minute with 30-mL portions of hexanesaturated acetonitrile.
11.2.2 Combine and transfer the acetonitrile phases to a l-L separatory funnel and add 650 mL
of reagent water and 40 mL of saturated sodium chloride solution. Mix thoroughly for
30 to 45 seconds. Extract with two 100-mi. portions of hexane by vigorously shaking
for 15 seconds.
11.2.3 Combine the hexane extracts in a 1-L separatory funnel and wash with two 100-mL
portions of reagent water. Discard the water layer and pour the hexane layer through a
drying column containing 7 to 10 cm of anhydrous sodium sulfate into a 500 -mL K-D
flask equipped with a 10-mL concentrator tube. Rinse the separatory funnel and column
with three 10-mL portions of hexane.
11.2.4 Concentrate the extracts to 6 to 10 mL in the K-D as directed in Section 10.6. Adjust
the extract volume to 10 mL with hexane.
11.2.5 Analyze by gas chromatography unless a need for further cleanup is indicated.
11.3 Florisil colun n cleanup: The following Florisil column cleanup procedure has been demonstrated
to be applicable to the seven organophosphorus pesticides listed in Table 3. It should also be
applicable to the cleanup of extracts for ethion.
11.3.1
Add a weight of Florisil (nominally 20 g) predetermined by calibration (Section 7.4 and
75) to a chromatographic column. Settle the Florisil by tapping the column. Add
anhydroussodiumsulfatetothetopoftheFlorisiltoformalayerlto 2 cmdeep. Add
60 mL of hexane to wet and rinse the sodium sulfate and Florisil. Just prior to exposure
of the sodium sulfate to air, stop the elution of the hexane by closing the stopcock on the
41rnm atnar9nhv column. Discard the eluate.
11.3.2 Adjust the sample extract volume to 10 mL with hexane and transfer it from the K-D
concentrator tube to the Florisil column. Rinse the tube twice with 1 to 2 mL hexane,
adding each rinse to the column.
11.3.3 Place a 500-mL K D flask and clean concentrator tube under the chromatography
column. Drain the column into the flask until the sodium sulfate layer is nearly exposed.
Elute the column with 200 mL of 6% ethyl ether in hexane (WV) (Fraction 1) using a
drip rate of about 5 mL/min. Remove the K-D flask and set aside for later
concentration. Elute the column again, using 200 mL of 15% ethyl ether in hexane
(WV) (Fraction 2) into a second K-D flask. Perform a third elution using 200 mL of
50% ethyl ether in hexane (WV) (Fraction 3) and a final elution with 200 mL of 100%
ethyl ether (Fraction 4) into separate K-I) flasks. The elution patterns for seven of the
pesticides are shown in Table 3.
11.3.4 Concentrate the eluates by standard K-D techniques (Section 10.6), using the water bath
at about 85°C (75°C for Fraction 4). Adjust final volume to 10 mL with hexane.
Analyze by gas chromatography.
70
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Method 614
11.4 Removal of sulfur: 9 Elemental sulfur will elute in Fraction 1 of the Florisil cleanup procedure.
If a large amount of sulfur is present in the extract, it may elute in all fractions. If so, each
fraction must be further treated to remove the sulfur.
1 1.4.1 Add one or two boiling chips to the l0-mL hexane solution contained in a concentrator
tube. Attach a micro-Snyder column and concentrate the extract to about 0.2 mL in a
hot water bath at 85°C. Remove the micro K-D from the bath, cool, and adjust the
volume to 0.5 mL with hexane.
11.4.2 Plug a disposable pipette with a small quantity of glass wool. Add enough alumina to
produce a 3-cm column after settling. Top the alumina with a 0.5-cm layer of anhydrous
sodium sulfate.
11.4.3 Quantitatively transfer the concentrated extract to the alumina microcolumn using a
100-FL syringe. Rinse the ampul with 200 p L of hexane and add to the microcolumn.
11.4.4 Elute the microcolumn with 3 mL of hexane and discard the eluate.
11.4.5 Elute the column with 5 mL of 10% hexane in methylene chloride, and collect the eluate
in a l0-mL concentrator tube. Adjust final volume to 10 niL with hexane. Analyze by
gas chromatography.
12. GAs CHROMATOGRAPHY
12.1 Table 1 summarizes the recommended operating conditions for the gas chromatograph. Included
in this table are estimated retention-times and method detection limits that can be achieved by this
method. Other packed columns, chromatographic conditions, or detectors may be used if the
requirements of Section 8.2 are met. Capillary (open-tubular) columns may also be used if the
relative standard deviations of responses for replicate injections are demonstrated to be less
than 6% and the requirements of Section 8.2 are met.
12.2 Calibrate the system daily as described in Section 7.
12.3 If the internal standard approach is being used, add the internal standard to sample extracts
immediately before injection into the instrument. Mix thoroughly.
12.4 Inject 1 to 5 L of the sample extract using the solvent-flush technique.’° Record the volume
injected to the nearest 0.05 L, and the resulting peak size in area or peak height units. An
automated system that consistently injects a constant volume of extract may also be used.
12.5 The width of the retention-time window used to make identifications should be based upon
measurements of actual retention-time variations of standards over the course of a day. Three
times the standard deviation of a retention time can be used to calculate a suggested window size
for a compound. However, the experience of the analyst should weigh heavily in the
interpretation of chromatograms.
12.6 If the response for the peak exceeds the working range of the system, dilute the extract and
reanalyze.
12.7 If the measurement of the peak response is prevented by the presence of interferences, further
cleanup is required.
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Method 614
13. CALCULATIONS
13.1 Determine the concentration of individual compounds in the sample.
13.1.1 If the external standard calibration procedure is used, calculate the amount of material
injected from the peak response using the calibration curve or calibration factor in
Section 7.2.2. The concentration in the sample can be calculated as follows:
Equation 2
Concentration, vigIL = ______
(V)(V,)
where
A = Amount of material injected, in ng
V = Volume of extract injected, in 1 iL
V = Volume of total extract, in pL
V 1 = Volume of water extracted, in mL
13.1.2 If the internal standard calibration procedure was used, calculate the concentration in the
sample using the response factor (RF) determined in Section 7.3.2 as follows:
Equation 3
(A)(I)
Concentration, ig/L =
where
.
A, = Response for parameter to be measured
A,, = Response jbr the internal standard
I , = Amount of internal standard added to each extract,
in
pg
V = Volume of ter extracted, in L
13.2 Report results in microgtauis per liter without correction for recovery data. When duplicate and
spiked samples are analyzed, report all data Obtained with the sample results.
13.3 For samples processed as part of a set where the laboratory spiked sample recovery falls outside
of the control limits in Section 8.3, data for the affected parameters must be labeled as suspect.
14. GC/MS CONRRMA lION
14.1 It is recommended that GC/MS techniques be judiciously employed to support qualitative
compound identifications made with this method. The mass spectrometer should be capable of
scanning the mass range from 35 amu to a mass 50 amu above the molecular weight of the
compound. The instrument must be capable of scanning the mass range at a rate to produce at
least 5 scans per peak but not to exceed 7 seconds per scan utilizing a 70 V (nominal) electron
energy in the electron impact ionization mode. A GC to MS interface constructed of all glass or
glass-lined materials is recommended. A computer system should be interfaced to the mass
72
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Method 614
spectrometer that allows the continuous acquisition and storage on machine-readable media of all
mass spectra obtained throughout the duration of the chromatographic program.
14.2 Gas chromatographic columns and conditions should be selected for optimum separation and
performance. The conditions selected must be compatible with standard GC/MS operating
practices. Chromatographic tailing factors of less than 5.0 must be achieved.”
14.3 At the beginning of each day that confirmatory analyses are to be performed, the GC/MS system
must be checked to see that all decafluorotriphenyl phosphine (DFTPP) performance criteria are
achieved. 12
14.4 To confirm an identification of a compound, the background-corrected mass spectrum of the
compound must be obtained from the sample extract and compared with a mass spectrum from
a stock or calibration standard analyzed under the same chromatographic conditions. It is
recommended that at least 25 ng of material be injected into the GC/MS. The criteria below must
be met for qualitative confirmation.
14.4.1 All ions that are present above 10% relative abundance in the mass spectrum of the
standard must be present in the mass spectrum of the sample with agreement to plus or
minus 10%. For example, if the relative abundance of an ion is 30% in the mass
spectrum of the standard, the allowable limits for the relative abundance of that ion in
the mass spectrum for the sample would be 20% to 40%.
14.4.2 The retention-time of the compound in the sample must be within 6 seconds of the same
compound in the standard solution.
14.4.3 Compounds that have very similar mass spectra can be explicitly identified by GC/MS
only on the basis of retention-time data.
14.5 Where available, chemical ionization mass spectra may be employed to aid in the qualitative
identification process.
14.6 Should these MS procedures fail to provide satisfactory results, additional steps may be taken
before reanalysis. These may include the use of alternate packed or capillary GC columns or
additional cleanup (Section 11).
15. METHOD PERFORMANCE
15.1 The method detection limit (MDL) is defined as the minimum concentration of a substance that
can be measured and reported with 99% confidence that the value is above zero.’ 3 The MDL
concentrations listed in Table 1 were obtained using reagent water.’ 4
15.2 In a single laboratory, Susquehanna University, using spiked tap water samples, the average
recoveries presented in Table 3 were obtained. The standard deviation of the percent recovery
is also included in Table 3•14
73
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Method 614
References
1. Methods for Benzidine, Chlorinated Organic Compounds, Pentachiorophenol and Pesticides in
Water and Wastewater,” U.S. Environmental Protection Agency, Environmental Monitoring and
Support Laboratory - Cincinnati, Ohio, September 1978.
2. ASTM Annual Book of Standards, Part 31, D3694, “Standard Practice for Preparation of Sample
Containers and for Preservation, “American Society for Testing and Materials, Philadelphia, PA,
p. 679, 1980.
3. “Carcinogens - Working with Carcinogens,” Department of Health, Education, and Welfare,
Public Health Service, Center for Disease Control, National Institute for Occupational Safety and
Health, Publication No. 77-206, Aug. 1977.
4. M () 1Ifr Safety and Health Standards, General Industry,” (29 CFR 1910), Occupational Safety and
Health Administration, OSHA 2206, (Revised, January 1976).
5. “Safety in Academic Chemistry Laboratories,” American Chemical Society Publication,
Committee on Chemical Safety, 3rd Edition, 1979.
6. ASTM Annual Book of Standards, Part 31, D3086, Appendix X3, “Standardization of Florisil
Column by Weight Adjustment Based on Adsorption of Lauric Acid,” American Society for
Testing and Materials, Philadelphia, PA, p 765, 1980.
7. “Handbook for Analytical Quality Control in Water and Wastewater Laboratories,”
EPA-600/4-79-019, U. S. Environmental Protection Agency, Environmental Monitoring and
Support Laboratory - Cincinnati, Ohio, March 1979.
8. ASTM Annual Book of Standards, Part 31, D3370, “Standard Practice for Sampling Water,”
American Society for Testing and Materials, Philadelphia, PA, p. 76, 1980.
9. Law, L. M. and D. F. Goerlitz, “Mi Iwnn Chromatographic Cleanup for the Analysis of
Pesticides in Water,” Journoi of the Association of Official Analytical Chemists, 53, 1276, (1970).
10. Burke, 3. A., “Gas Chromatography for Pesticide Residue Analysis; Some Practical Aspects,”
Journal of the Association of Official Analytical Qiesnists. 48, 1037 (1965).
11. McNair, H.M. and Bonelli, E. 3., Basic Chromatography, Consolidated Printing, Berkeley,
California, p. 52, 1969.
12. Eichelberger, J.W., Harris, L.E., and Budde, W.L. “Reference Compound to Calibrate Ion
Abundance Measurement in Gas Chromatography-Mass Spectrometry,” Analytical Ozemistiy, 47,
995 (1975).
13. Glaser, l.A. etal, “Trace Analysis for Wastewaters,” Ein ronmentd Science & Technology, 15,
1426(1981).
74
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Method 614
References (cont.)
14. McGrath, T. F., “Recovery Studies of Pesticides From Surface and Drinking Waters,” Final
Report for U.S. EPA Grant R804294, Environmental Monitoring and Support Laboratory,
Cincinnati, Ohio.
75
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Method 614
Table 1. Chromatographic Conditions and Method Detection Limits
Pa,ameter Retention lime Method
(minutes) Detection
______________ Limit
Column 1 Column 2 (pg/LI
Diazinon 1.8 1.8 0.012
Disulfoton 1.9 2.1 ND
Demeton 2.3 2.1 ND
Parathion 2.5 3.7 0.012
methyl
Malathion 2.9 3.9 ND
Parathion ethyl 3.1 4.5 0.012
Ethion 6.8 9.1 ND
Azinphos 14.5 29.9 ND
methyl
ND = Not determined
Column 1 conditions: Gas-Chrom Q (100/120 mesh) coated with 3% OV-l packed in a 1.8 m long by
4 mm ID glass column with nitrogen carrier gas at a flow rate of 60 mL/min. Column temperature,
isothermal at 200°C. A flame photometric detector was used with this column to determine the MDL.
Column 2 conditions: Gas Chrom Q (100/120 mesh) coated with 1.5% OV- 17+l.95% QF-l packed in
a 1.8 m long by 4 mm II) glass column with nitrogen carrier gas at 70 mL/min. flow rate. Column
temperature, isothermal at 2 12°C.
76
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Method 614
Table 2. Single-Operator Accuracy and Precision
Parameter Average Standard Spike Number
Percent Deviation Range of Matrix
Recovery (96) (pg/L) Analyses Types
fliazinon 94 5.2 0.04-40 27 4
Parathion methyl 95 3.2 0.06-60 27 4
Parathion ethyl 102 4.1 0.07-70 27 4
Table 3. Florisil Fractionation Patterns and Acetonitrile Partition Applicability
Percent Recovery by Fraction Acetonitrile
_____________________________________ Partition
Parameter No. 1 No. 2 No. 3 No. 4 Applicability
Demeton 100 ND
Disulfoton 100 ND
Diazinon 100 Yes
Malathion 5 95 y
Parathion ethyl 100 Yes
Parathion methyl 100 Yes
Azinphos methyl 20 80 ND
Ethion ND ND ND ND Yes
ND = Not determined
Florisil eluate composition by fraction
Fraction 1 - 200 mL of 6% ethyl ether in hexane
Fraction 2 - 200 mL of 15% ethyl ether in hexane
Fraction 3 - 200 mL of 50% ethyl ether in hexane
Fraction 4 - 200 mL of ethyl ether
77
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Method 614.1
The Determination of
Orgénophosphorus Pesticides in
Municipal and Industrial
Waste water
-------
Method 614.1
The Determination of Organophosphorus
Pesticides in Municipal and Industrial Waste water
1. SCOPE AND APPLICA TION
1.1 This method covers the determination of certain organophosphorus pesticides in municipal and
industrial wastewater. The following parameters may be determined by this method.
Parameter S TO RET No. CA S No.
Dioxathion 78-34-2
EPN 2104-64-5
Ethion 39398 563-12-2
Terbufos 13071-79-9
1.2 The estimated detection limit (EDL) for each parameter is listed in Table 1. The EDL was
calculated from the minimum detectable response of the nitrogen/phosphorus detector equal to 5
times the gas chromatographic (GC) background noise assuming a 1 .0-mL final extract volume
of a 1-L reagent water sample and an injection of 5 giL. The EDL for a specific wastewater may
be different depending on the nature of interferences in the sample matrix.
1.3 This is a gas chromatographic (GC) method applicable to the determination of the compounds
listed above in municipal and industrial discharges. When this method is used to analyze
unfamiliar samples for any or all of the compounds listed above, compound identifications should
be supported by at least one additional qualitative technique. Section 13 provides gas
chromatograph/mass spectrometer (GC/MS) conditions appropriate for the qualitative confirmation
of compound identifications.
1.4 This method is restricted to use by or under the supervision of analysts experienced in the
operation of gas chromatographs and in the interpretation of chromatograms.
2. SUMMARY OF METHOD
2.1 Organophosphorus pesticides are removed from the sample matrix by extraction with 15%
methylene chloride in hexane. The extract is dried, exchanged into hexane, and analyzed by gas
chromatography. Column chromatography is used as necessary to eliminate interferences which
may be encountered. Measurement of the pesticides is accomplished with a nitrogenlphosphorus-
specific detector.
2.2 Confirmatory analysis by GC/MS is recommended when a new or undefined sample type is being
analyzed if the concentration is adequate for such determination.
3. INTERFERENCES
3.1 Solvent, reagents, glassware, and other sample-processing hardware may yield discrete artifacts
and/or elevated baselines causing misinterpretation of gas chromatograms. All of these materials
81
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Method 614.1
must be demonstrated to be free from interferences under the conditions of the analysis by running
laboratory reagent blanks as described in Section 9.1.
3.1.1 The use of high-purity reagents and solvents helps to minimize interference problems.
Purification of solvents by distillation in all-glass systems may be required.
3.1.2 Glassware must be scrupulously cleaned.’ Clean all glassware as soon as possible after
use by rinsing with the last solvent used in it. This should be followed by detergent
washing with hot water and rinses with tap water and reagent water. It should then be
drained dry and heated in a muffle furnace at 400°C for 15 to 30 minutes. Solvent rinses
with acetone and pesticide-quality hexane may be substituted for the heating. Volumetric
ware should not be heated in a muffle furnace. After drying and cooling, glassware
should be sealed and stored in a clean environment to prevent any accumulation of dust
or other contaminants. Store the glassware inverted or capped with aluminum foil.
3.2 Interferences coextracted from the samples will vary considerably from source to source,
depending on the diversity of the industrial complex or municipality being sampled. While
general cleanup procedures are provided as part of this method, unique samples may require
additional cleanup approaches to achieve the detection limits listed in Table 1.
4. SAFETY
4.1 The toxicity Qr carcinogenicity of each reagent used in this method has not been precisely defined;
however, each chemical compound should be treated as a potential health hazard. From this
viewpoint, exposure to these chemicals must be reduced to the lowest possible level by whatever
means available. The laboratory is responsible for maintaining a current awareness file of OSHA
regulations regarding the safe handling of the chemicals specified in this method. A reference file
of material data handling sheets should also be made available to all personnel involved in the
chemical analysis. Additional references to laboratory safety are available and have been
identifled for the information of the analyst.
5. APPARA TUS AND EQUIPMENT
5.1 Sample containers: Narrow-mouth glass bottles, 1-L or 1-quart volume, equipped with
polytetrafluoroethylene (PTFE)-lined screw-caps. Wide-mouth glass bottles, 1-quart volume,
equipped with FrFE-lined screw-caps may also be used. Prior to use, wash bottles and cap liners
with detergent and rinse with tap and distilled water. Allow the bottles and cap liners to air dry,
then muffle at 400°C for 1 hour. After cooling, rinse the cap liners with hexane, seal the bottles,
and store in a dust-free environment.
5.1.1 Automatic sampler (optional): Must incorporate glass sample containers for the
collection of a minimum of 250 mL. Sample containers must be kept refrigerated at 4°C
and protected from light during compositing. If the sampler uses a peristaltic pump, a
minimum length of compressible silicone rubber tubing may be used. Before use,
however, the compressible tubing should be thoroughly rinsed with methanol, followed
by repeated rinsings with distilled water to minimize the potential for contamination of
the sample. An integrating flow meter is required to collect flow-proportional
composites.
82
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Method 614.1
5.2 Kuderna-danish (K-D) glassware.
5.2.1 Synder column: Three-ball macro (Kontes K-503000-0121 or equivalent) and two-ball
micro (Kontes K-569001-0219 or equivalent).
5.2.2 Concentrator tube: 10-mL, graduated (Kontes K-570050-1025 or equivalent) with
ground-glass stopper.
5.2.3 Evaporative flask: 500-mL (Kontes K-570001-0500 or equivalent). Attach to
concentrator tube with springs.
5.3 Gas chromatography system.
5.3.1 The gas chromatograph must be equipped with a glass-lined injection port compatible
with the detector to be used. A data system is recommended for measuring peak areas.
5.3.1.1 Column 1: 180 cm long by 2 mm ID, glass, packed with 3% OV-225 on
Supelcoport (100/120 mesh) or equivalent.
5.3.1.2 Column 2: 120 cm long by 2 mm ID, PyrexR glass, packed with 1.5%
OV-17/1 .95 % QF-1 on Gas Chrom Q, 80/100 mesh or equivalent.
5.3.1.3 Column 1 was used to develop the accuracy and precision statements in
Section 12. Guidelines for the use of alternative column packings are
provided in Section 10.3.1.
5.3.1.4 Detector: nitrogen/phosphorus. This detector has proven effective in the
analysis of wastewaters for the parameters listed in the scope and was used
to develop the method performance statements in Section 12. Guidelines for
the use of alternative detectors are provided in Section 10.3.1.
5.4 Chromatographic column: 300 mm long by 10 mm 11) Chromafiex, equipped with coarse-fitted
bottom plate and PTFE stopcock. (Kontes K-420540-0213 or equivalent).
5.5 Drying column: Approximately 400 mm long by 20 mm ID borosilicate glass, equipped with
coarse-fitted bottom plate.
5.6 Miscellaneous.
5.6.1 Balance: Analytical, capable of accurately weighing to the nearest 0.0001 g.
5.6.2 Separatory funnel: 2-L, equipped with PTFE stopcock.
5.6.3 Water bath: Heated with concentric ring cover, capable of temperature control (±2°C).
The bath should be used in a hood.
5.6.4 Standard solution storage containers: 15-mL bottles with PTFE-lined screw-caps.
5.6.5 Boiling chips: Approximately 10/40 mesh. Heat to 400°C for 30 minutes or perform
a Soxhlet extraction with methylene chloride.
6. REAGENTS AND CONSUMABLE MA TERIALS
6.1 Reagents.
6.1.1 Acetone, hexane, and methylene chloride: Demonstrated to be free of analytes.
6.1.2 Silica gel: Woelm 70-230 mesh. Activate approximately 100 g of silica gel at 200°C
for 6 hours in a tared 500-mL Erlenmeyer flask with ground-glass stopper. Allow to
83
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Method 614.1
cool to room temperature, reweigh, and determine the weight of activated silica gel.
Deactivate by adding 3% by weight of distilled water. Restopper the flask, and shake
on a wrist-action shaker for at least 1 hour. Allow to equilibrate for 3 or more hours at
room temperature.
6.1.3 Reagent water: Reagent water is defined as a water in which an interferent is not
observed at the method detection limit of each parameter of interest.
6.1.4 Sodium hydroxide (NaOH) solution (iON): Dissolve 40 g NaOH in reagent water and
dilute to iOOmL.
6.1.5 Sodium sulfate: Granular, anhydrous. Condition by heating at 400°C for 4 hours in a
shallow tray.
6.1.6 Sulfuric acid (H 2 S0 4 ) solution (1+1): Add measured volume of concentrated H 2 S0 4 to
equal volume of reagent water.
6.2 Standard stock solutions (1.00 gI tL): These solutions may be purchased as certified solutions
or prepared from pure standard materials using the following procedures.
6.2.1 Prepare standard stock solutions by accurately weighing about 0.0100 g of pure material.
Dissolve the material in bexane and dilute to volume in a 10-mL volumetric flask.
Larger volumes can be used at the convenience of the analyst. If compound purity is
certified at 96% or greater, the weight can be used without correction to calculate the
concentration of the standard stock.
6.2.2 Store standard stock solutions at 4°C in 15-mL bottles equipped with PTFE-lined
screw-caps. Standard stock solutions should be checked frequently for signs of
degradation or evaporation, especially just prior to preparing calibration standards from
them.
6.2.3 Standard stock solutions must be replaced after 6 months, or sooner if comparison with
check standards indicates a problem.
7. SAMPLE COLLECTION, PRESERVATiON. AND STORAGE
7.1 Collect all samples in duplicate. Grab samples must be collected in glass containers.
Conventional sampling practices should be followed, except that the bottle must not be prewashed
with sample before collection.
7.2 The s2mples must be iced or refrigerated at 4°C from the time of collection until extraction.
7.3 Chemical preservatives should not be used in the field unless more than 24 hours will elapse
before delivery to the laboratory. If the samples will not be extracted within 48 hours of
collection, the sample should be adjusted to a pH range of 6.0 to 8.0 with sodium hydroxide or
sulfuric acid.
7.4 All samples must be extracted within 7 days and completely analyzed within 40 days of
extraction. 6
84
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Method 614.1
8. CALIBRA TION AND STANDARDIZA T1ON
A set of at least three calibration solutions containing the method analytes is needed.
One calibration solution should contain each analyte at a concentration approaching but
greater than the estimated detection limit (Table 1) for that compound; the other two
solutions should contain analytes at concentrations that bracket the range expected in
samples. For example, if the detection, limit for a particular analyte is 0.2 igIL, and a
sample expected to contain approximately 5 g/L is analyzed, solutions of standards
should be prepared at concentrations of 0.3 g/L, 5 g/L, and 10 ig/L for the particular
analyte.
8.1.2 To prepare a calibration solution, add an appropriate volume of a standard stock solution
to a volumetric flask and dilute to volume with hexane.
8.1.3 Starting with the standard of lowest concentration, analyze each calibration standard
according to Section 10.3.2 and tabulate peak height or area responses versus the mass
of analyte injected. The results can be used to prepare a calibration curve for each
compound. Alternatively, if the ratio of response to concentration (calibration factor) is
a constant over the working range (<10% relative standard deviation), linearity through
the origin can be assumed and the average ratio or calibration factor can be used in place
of a calibration curve.
8.1.4 The working calibration curve or calibration factor must be verified on each working day
by the measurement of one or more calibration standards. If the response for any analyte
varies from the predicted response by more than ± 10%, the test must be repeated using
a fresh calibration standard. If the results still do not agree, generate a new calibration
curve.
9. QUALITY CONTROL
9.1 Monitoring for interferences: Analyze a laboratory reagent blank each time a set of samples is
extracted. A laboratory reagent blank is a 1-L aliquot of reagent water. If the reagent blank
contains a reportable level of any analyte, immediately check the entire analytical system to locate
and correct for possible interferences and repeat the test.
9.2 Assessing accuracy.
9.2.1 After every 10 samples, and preferably in the middle of each day, analyze a laboratory
control standard. Calibration standards may not be used for accuracy assessments and
the laboratory control standard may not be used for calibration of the analytical system.
9.2.1.1 Laboratory control standard concentrate: From stock standards prepared as
described in Section 6.2, prepare a laboratory control standard concentrate
that contains each analyte of interest at a concentration of 2 ig/ml in acetone
or other suitable solvent. 7
9.2.1.2 Laboratory control standard: Using a pipette, add 1.00 mL of the laboratory
control standard concentrate to a 1-L aliquot of reagent water.
8.1 Calibration.
8.1.1
85
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Method 614.1
92.1.3 Analyze the laboratory control standard as described in Section 10. For each
analyte in the laboratory control standard, calculate the percent recovery (P,)
with the equation:
Equation 1
P =
T,
n*ere
Si = The analytical results from the laboratory control standard, in g/L
= The baoMl concentration of the spike, in gIL
9.2.2 At least annually, the laboratory should participate in formal performance evaluation
studies, where solutions of unknown concentrations are analyzed and the performance of
all participants is compared.
9.3
Assessing precision.
9.3.1 PreCisiOn assessments for this method are based upon the analysis of field duplicates
(Section 7.1). Analyze both sample bottles for at least 10% of all samples. To the extent
practical, the samples for duplication should contain reportable levels of most of the
analytes.
9.3.2 For each analyte in each duplicate pair, calculate the relative range’ (RR ) with the
equation:
Equation 2
RR 1 = 100R 1
R 1 = The absolute difference betwwt the duplicate nsea.cwesnents I , and X,, In g/L
X - The awrage concentration found ‘ 2 , In igIL
9.3.3 Individual relative range measurements are pooled to determine average relative range
or to develop an expression of relative range as a function of concentration.
10. PROCEDURE
10.1 Sample extraction.
10.1.1 Mark the water meniscus on the side of the sample bottle for later determination of
sknlple volume. Pour the entire sample into a 2-L separatory funnel. Check the pH of
the sample with wide-range pH paper and adjust to within the range of 5 to 9 with
sodium hydroxide or sulfuric acid.
86
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Method 614.1
10.1.2 Add 60 mL of 15% methylene chloridelhexane to the sample bottle and shake for 30
seconds to rinse the walls. Transfer the solvent to the separatory funnel and extract the
sample by shaking the funnel for 2 minutes with periodic venting to release vapor
pressure. Allow the organic layer to separate from the water phase for a minimum of
10 minutes. If the emulsion interface between layers is more than one-third thevolume
of the solvent layer, the analyst must employ mechanical techniques to complete the
phase separation. The optimum technique depends on the sample, but may include
stirring, filtration of the emulsion through glass wool, or centrifugation. Collect the
extract in a 250-mL Erlenmeyer flask.
10.1.3 Add an additional 60-mL volume of 15% methylene chloridefhexane to the sample bottle
and complete the extraction procedure a second time, combining the extracts in the
Erlemneyer flask.
10.1.4 Perform a third extraction in the same manner. Pour the combined extract through a
drying column containing about 10 cm of anhydrous sodium sulfate, and collect it in a
500-mL K-D flask equipped with a 10-mL concentrator tube. Rinse the Erlenmeyer flask
and column with 20 to 30 mL of methylene chloride to complete the quantitative transfer.
10.1.5 Add one or two clean boiling chips to the flask and attach a three-ball Snyder column.
Prewet the Snyder column by adding about 1 rnL of methylene chloride to the top. Place
the K-D apparatus on a hot water bath (80 to 85°C) so that the concentrator tube is
partially immersed in the hot water and the entire lower rounded surface of the flask is
bathed in steam. Adjust the vertical position of the apparatus and the water temperature
as required to complete the concentration in 15 to 20 minutes. At the proper rate of
distillation, the balls of the column will actively chatter but the chambers will not flood.
When the apparent volume of liquid reaches 1 mL, remove the K-D apparatus and allow
it to drain and cool for at least 10 minutes. Remove the Snyder column and rinse the
flask and its lower joint into the concentrator tube with 1 to 2 mL of hexane. A 5-mL
syringe is recommended for this operation. If the extract requires cleanup, proceed to
Section 10.2 (cleanup and separation). If cleanup has been performed or if the extract
does not require cleanup, proceed with Section 10.1.6.
10.1.6 Add a clean boiling chip to the concentrator tube. Attach a two-ball micro-Snyder
column. Prewet the micro-Snyder column by adding about 0.5 mL of hexane to the top.
Place this micro K-D apparatus on a steaming-water bath (80 to 85°C) so that the
concentrator tube is partially immersed in the hot water. Adjust the vertical position of
the apparatus and water temperature as required to complete the concentration in 5 to 10
minutes. At the proper rate of distillation, the balls will actively chatter but the chambers
will not flood. When the apparent volume of liquid reaches 0.5 mL, remove the K-D
apparatus and allow it to drain and cool for at least 10 minutes. Remove the
micro-Snyder column and rinse its lower joint into the concentrator tube with a small
volume of hexane. Adjust the final volume to 1.0 mL or to a volume suitable for
cleanup or gas chromatography, and stopper the concentrator tube; store refrigerated if
further processing will not be performed immediately. If the extracts will be stored
longer than 2 days, they should be transferred to PTFE-sealed screw-cap bottles.
Proceed with gas chromatographic analysis.
87
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Method 614.1
10.1.7 Determine the original sample volume by refilling the sample bottle to the mark and
transferring the liquid to a 1,000-mL graduated cylInder. Record the sample volume to
the nearest 5 mL.
10.2 Cleanup and separation.
10.2.1 Cleanup procedures may not be necessary for a relatively clean sample matrix. The
cleanup procedures recommended in this method have been used for the analysis of
various clean waters and municipal effluents. The sIlica gel procedure allows for a select
fractionation of the compounds and will eliminate non-polar materials. The
single-operator precision and accuracy data in Table 2 were gathered using the
recommended cleanup procedures. If particular circumstances demand the use of an
alternative cleanup procedure, the analyst must determine the elution profile and
demonstrate that the recovery of each compound of interest is no less than that recorded
in Table 2.
10.2.2 Prepare silica gel columns using a glass column 200 mm/ng by 10 mm ID. Rinse
column with hexane. Add approximately 50 mL of hexane to the empty column. Add
3.5 grams of 3% deactivated silica geL Pack by rotating slowly to release air bubbles.
Top with 1.5 cm of Na 2 SO 4 . Drain hexane to the top of Na 2 SO 4 layer.
10.2.3 Just prior to exposure of the sodium sulfate layer to the air, transfer the sample extract
onto the column using an additional 2 mL of hexane to complete the transfer.
10.2.4 Just prior to exposure of the sodium sulfate layer to the air, add 30 mL of 6% methylene
chioride/hexane and continue the elution of the column, collecting the eluate in a 500-mL
K-D flask equipped with a 10-niL concentration tube. Elution of the column should be
at a rate of about2 niL per minute. Add 50 mL of hexane to the flask and concentrate
the collected fraction by the standard technique prescribed in Sections 10.1.5 and 10.1.6.
10.2.5 Continue the elution of the column according to the scheme outlined in Table 3. The
elution of the compounds may vary with different sample matrices.
10.2.6 Analyze the fractions by gas chromatography.
10.3 Gas chromnatographic analysis.
10.3.1 Recommended columftc and detector for the gas chromatography system are described
in Section 5.3.1. Table 1 summarizes the recommended operating conditions for the gas
chromatograph. Included In this table are estimated retention times and detection limits
that can be achieved by this method. Examples of the separations achieved are shown
in Figures 1 and 2. Other packed columns, chromatographic conditions, or detectors
may be used if data quality comparable to Table 2 are achieved. Capillary (open-tubular)
columns may also be used if the relative standard deviations of responses for replicate
injections are demonstrated to be less than 6% and data quality comparable to Table 2
are achieved.
10.3.2 Inject 2 to 5 p1. of the sample extract using the solvent-flush technique.’ Record the
volume injected to the nearest 0.05 giL, the total extract volume, and the resulting peak
size in area or peak height units.
10.3.3 The width of the retention-time window used to make identifications should be based
upon measurements of actual retention-time variations of standards over the course of the
88
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Method 614.1
day. Three times the standard deviation of a retention time for a compound can be used
to calculate a suggested window size; however, the experience of the analyst should
weigh heavily in the interpretation of chromatograms.
10.3.4 If the response for the peak exceeds the working range of the system, dilute the extract
and reanalyze.
10.3.5 If the measurement of the peak response is prevented by the presence of interferences,
further cleanup is required.
11. CALCULATIONS
11.1 Determine the concentration (C) of individual compounds in the sample in micrograms per liter
with the equation:
Equation 3
(A)(V)
Concentration, gIL = (V)(V)
.
where
A = Amount of material injected, in ng
V 1 = Voiwne of extract injected, in pL
= Volume of total extract, in huL
Vs = Volume of water extracted, in mL
11.2 Report the results for the unknown samples in microgram per Liter. Round off the results to the
nearest 0.1 g/L or two significant figures.
12. METHOD PERFORMANCE
12.1 Estimated detection limits (EDL) and associated chromatographic conditions are listed in Table 1.
The detection limits were calculated from the minimum detectable response of the N/P detector
equal to 5 times the GC background noise, assuming a 1.O-mL final extract volume of a l-L
sample and a GC injection of 5 pL.
12.2 Single laboratory accuracy and precision studies were conducted by ESE, 6 using spiked relevant
industrial wastewater samples. The results of these studies are presented in Table 2.
13. GC/MS CONFIRMATION
13.1 It is recomended that GCIMS techniques be judiciously employed to support qualitative
identifications made with this method. The mass spectrometer should be capable of scanning the
mass range from 35 amu to a mass 50 amu above the molecular weight of the compounds of
interest. The instrument must be capable of scanning the mass range at a rate to produce at least
5 scans per peak, but not to exceed 7 scans per peak utilizing a 70 V (nominal) electron energy
in the electron impact ionization mode. A GC to MS interface constructed of all-glass or
glass-lined materials is recommended. A computer system should be interfaced to the mass
89
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Method 614.7
spectrometer that allows the continuous acquisition and storage on machine-readable media of all
mass spectra obtained throughout the duration of the chromatographic program.
13.2 Gas chromatographic columns and conditions should be selected for optimum separation and
performance. The conditions selected must be compatible with standard GCIMS operating
practIces. Chromatographic tailing factors of less than 5.0 must be achieved. The calculation of
tailing factors is illustrated in Method 625.’°
13.3 Atthebeginningofeachdaythatconflrmatoryanalysesaretobeperformed, theGCIMS system
must be checked to see that all DFTPP performance criteria are achieved. 1 ’
13.4 To confirm an identification of a compound, the background-corrected mass spectrum of the
compound must be obtained from the sample extract and compared with a mass spectrum from
a stock or calibration standard analyzed under the same chromatographic conditions. It is
recommended that at least 25 ng of material be injected into the GC/MS. The criteria below must
be met for qualitative confirmation.
13.4.1 The molecular ion and other ions that are present above 10% relative abundance in the
mass spectrum of the standard must be present in the mass spectrum of the sample with
agreement to ± 10%. For example, if the relative abundance of an ion is 30% in the
mass spectrum of the standard, the allowable limits for the relative abundance of that ion
in the mass spectrum forthe samplewould be20to 40%.
13.4.2 The i etention-time of the compound in the sample must be within 6 seconds of the same
compound in the standatd solution.
13.4.3 Compounds that have similar mass spectra can be explicitly identified by GC/MS only
on the basis of retention-time data.
13.5 Where available, chemical ionization mass spectra may be employed to aid in the qualitative
identification process.
13.6 Should these MS procedures fail to provide satisfactory results, additional steps may be taken
before reanalysis. These may include the use of alternative packed or capillary GC columns or
additional cleanup.
90
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Method 614.1
References
1. ASTM Annual Book of Standards, Part 31, D3694, “Standard Practice for Preparation of Sample
Containers and for Preservation,” American Society for Testing and Materials, Philadelphia, PA,
p. 679, 1980.
2. “Carcinogens - Working with Carcinogens,” Department of Health, Education, and Welfare,
Public Health Service, Center for Disease Control, National Institute for Occupational Safety and
Health, Publication No. 77-206, Aug. 1977.
3. “OSHA Safety and Health Standards, General Industry” (29 CFR 1910), Occupational Safety and
Health Administration, OSHA 2206 (Revised, January 1976).
4. “Safety in Academic Chemistry Laboratories,” American Chemical Society Publication,
Committee on Chemical Safety, 3rd Edition, 1979.
5. ASTM Annual Book of Standards, Part 31, D3370, “Standard Practice for Sampling Water,”
American Society for Testing and Materials, Philadelphia, PA, p. 76, 1980.
6. Test procedures for Pesticides iii Wastewaters, EPA Contract Report #68-03-2897. Unpublished
report available from U.S. Environmental Protection Agency, Environmental Monitoring and
Support Laboratory, Cincinnati, Ohio.
7. “Handbook for Analytical Quality Control in Water and Wastewater Laboratories,”
EPA-600/4-79-019, U.S. Environmental Protection Agency, Environmental Monitoring and
Support Laboratory Cincinnati, Ohio 45268, March 1979.
8. Burke, J.A., “Gas Chromatography for Pesticide Residue Analysis; Some Practical Aspects,”
Journal of the Association of Official Analytical Chemists, 48, 1037 (1965T)
9. “Evaluation of Ten Pesticide Methods,” U.S. Environmental Protection Agency, Contract No.
68-03-1760, Task No. 11, U.S. Environmental Monitoring and Support Laboratory, Cincinnati,
Ohio 45268.
10. “Methods for Organic Chemical Analysis of Municipal and Industrial Wastewater,”
EPA-600/4-82-057, U.S. Environmental Protection Agency, Environmental Monitoring and
Support Laboratory, Cincinnati, Ohio 45268.
11. Eichelberger, J.W., Harris, L.E., and Budde, W.L., Anal. Chem., 46, 1912 (1975).
91
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Method 614.1
Table 1. Gas Chromatography of Organophosphorus Pesticides
Retention Tin,. (minutes) Detection Limit
Paramete , Column 1 Column 2 (pg/Li
Terbufos 1.41 1.9 .004
Dioxathion 2.3 2.3 .01
Ethion 8.3 6.4 0.1
EPN 13.3 8.3 0.2
Column 1: 180 cm long by 2 mm ID, glass, packed with 3% OV-225 on 100/120 Supelcoport; nitrogen
carrier gas at a flow rate of 50 mL/min. Column temperature is 200°C for 2 minutes, then programmed
at 5°/mm to 240°C and held for 5 minutes.
Column 2: 120 cm long by 2 mm ID, Pyrex’ t glass, packed with 1.5% OC-17/1.95% QF-1 on 80/100
mesh (las Chrom Q or equivalent; nitrogen carrier gas at a flow rate of 30 mL/min. Column temperature
is 180°Cfor2minutes,thenprogrammedat8°/minto250°Candheldfor4minutes.
Table 2. Single-Laboratory Accuracy and Precision
Spike Average Standard
Mattix Range Number of Percent Deviation
Parameter Type* (pg/L) Repicates Recovery (%)
DIOXathIOn 1 1,978.0 7 94.3 19.9
1 19.8 7 99.0 27.5
EPN 1 1,293.0 7 96.1 6.1
Ethion 1 1,788.0 7 89.2 4.5
Terbufbs 1 15.1 7 101.0 12.4
1 1,508.0 7 95.0 3.4
*1 = Combined industrial wastewaters
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Method 614.1
Table 3. Silica Gel Cleanup of Organophosphorus Pesticides
Percent Recoveries
Silica Gel Fraction * Tethufos Dioxathion Ethion EPN
1 0 0 0.8 0
2 0 0 1.9 0
3 93.0 35.1 94.9 46.4
4 0 52.7 3.0 56.0
Total Percent Recoveries 93.0 87.8 101 102
* Fraction 1: 30 mL 6% MeCI 2 in hexane
Fraction 2 : 30 mL 15% MeC1 2 in hexane
Fraction 3 30 mL 50% MeC1 2 in hexane
Fraction 4: 30 mL 100% MeC1 2
93
-------
MWhod 614.1
0 2.0
4.0 6.0 8.0 10.0
Retention Time (minutes)
12.0 14.0 16.0
Figure 1. Gas Chromatogram of Organophosphorous Pesticides (Column 1).
/
/Ethion
/
/EPN
94
-------
Method 614.1
1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0 11.0 12.0
Retention Time (minutes)
Figure 2. Gas Chromatogram of Organophosphorous Pesticides (Column 2).
/
Ethion
/
EPN
/
Dioxathion
0
95
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Method 615
The Determination of
Chloriná ted Herbicides in
Municipal and Industrial
Waste water
-------
Method 615
The Determination of Chlorinated Herbicides in Municipal and
Industrial Wastewater
SCOPE AND APPLICATiON
1.1 This method covers the determination of certain chlorinated herbicides. The following parent
acids can be determined by this method:
Parameter STORETNo. CAS No.
2,4-1) 39736 94-75-7
Dalapon 75-99-0
2,4-DB 94-82-6
Dicamba 1918-00-9
Dich lorprop 120-36-5
Dinoseb 88-85-7
MCPA 94-74-6
MCPP - 7085-19-0
2,4,5-T 39740 93-76-5
2,4,5-TP 39760 93-72-1
1.2 This method is also applicable to the determination of salts and esters of these compounds. These
include, but are not limited to: the isobutyl and isooctyl esters of 2,4-D; the isobutyl and isooctyl
esters of 2,4-DB; the isooctyl ester of MCPA; and the isooctyl ester of 2,4,5-TP. The actual form
of each acid is not distinguished by this method. Results are calculated and reported for each
listed parameter as total free acid.
1.3 This is a gas chromatographic (GC) method applicable to the determination of the compounds
listed above in industrial and municipal discharges as provided under 40 CFR 136.1. Any
modification of this method beyond those expressly permitted shall .be considered a major
modification subject to application and approval of alternative test procedures under 40 CFR 136.4
and 136.5.
1.4 The method detection limit (MDL, defined in Section 15) for each parameter is listed in Table 1.
The MDL for a specific wastewater may differ from those listed, depending upon the nature of
interferences in the sample matrix.
1.5 This method is restricted to use by or under the supervision of analysts experienced in the use of
gas chromatography and in the interpretation of gas chromatograms. Each analyst must
demonstrate the ability to generate acceptable results with this method using the procedure
described in Section 8.2.
1.6 When this method is used to analyze unfamiliar samples for any or all of the compounds above,
compound identifications should be supported by at least one additional qualitative technique.
This method describes analytical conditions for alternative gas chromatographic columns that can
be used to confirm measurements made with the primary column. Section 15 provides gas
99
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Method 615
chromatograph/ mass spectrometer (GCIMS) criteria appropriate for the qualitative confirmation
of compound identifications.
2. SUMMARY OF METHOD
2.1 A measured volume of sample, approximately 1 L, is acidified. The acid herbicides and their
esters and salts are extracted with ethyl ether using a separatory funnel. The derivatives are
hydrolyzed with potassium hydroxide and extraneous organic material is removed by a solvent
wash. After acidification, the acids are extracted and converted to their methyl esters using
diazomethane as the derivatizing agent. Excess reagent is removed, and the esters are determined
by electron capture (EC) gas chromatography. 1
3. INTERFERENCES
3.1 Method interferences may be caused by contaminants in solvents, reagents, glassware, and other
sample-processing apparatus that lead to discrete artifacts or elevated baselines in gas
chromatograms. All reagents and apparatus must be routinely demonstrated to be free from
interferences under the conditions of the analysis by running laboratory reagent blanks as
described in Section 8.5.
3.1.1 Glassware must be scrupulously cleaned. 2 Clean all glassware as soon as possible after
use by thoroughly rinsing with the last solvent used in it. Follow by washing with hot
water and detergent and thorough rinsing with dilute acid, tap and reagent water. Drain
dry, and heat in an oven or muffle furnace at 400°C for 15 to 30 minutes. Do not heat
volumetric ware. Thermally stable materials, such as PCBs, may not be eliminated by
this treatment. Thorough rinsing with acetone and pesticide-quality hexane may be
substituted for the heating. After drying and cooling, seal and store glassware in a clean
environment to prevent any accumulation of dust or other contaminants. Store inverted
or capped with aluminum foil.
3.1.2 The use of high-purity reagents and solvents helps to minimize interference problems.
Purification of solvents by distillation in all-glass systems may be required.
3.2 The acid forms of the herbicides are strong organic acids, which react readily with alkaline
substances and can be lost during analysis. Glassware and glass wool must be acid-rinsed with
(1+9) hydrochloric acid and the sodium sulfate must be acidified with sulfuric acid prior to use
to avoid this possibility.
3.3 Organic acids and phenols, especially chlorinated compounds, cause the most direct interference
with the determination. Alkaline hydrolysis and subsequent extraction of the basic solution
remove many chlorinated hydrocarbons and phthalate esters that might otherwise interfere with
the electron capture analysis.
3.4 Matrix interferences may be caused by contaminants that are coextracted from the sample. The
extent of matrix interferences will vary considerably from source to source, depending upon the
nature and diversity of the industrial complex or municipality sampled. The cleanup procedure
in Section 11 can be used to overcome many of these interferences, but unique samples may
require additional cleanup approaches to achieve the MDL listed in Table 1.
700
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Method 615
4. SAFETY
4.1 The toxicity or carcinogenicity of each reagent used in this method has not been precisely defined;
however, each chemical compound must be treated as a potential health hazard. From this
viewpoint, exposure to these chemicals must be reduced to the lowest possible level by whatever
means available. The laboratory is responsible for maintaining a current awareness file of OSHA
regulations regarding the safe handling of the chemicals specified in this method. A reference file
of material data handling sheets should also be made available to all personnel involved in the
chemical analysis. Additional references to laboratory safety are available and have been
identified for the information of the analyst.
4.2 Diazomethane is a toxic carcinogen and can explode under certain conditions. The following
precautions must be followed:
4.2.1 Use only a well-ventilated hood; do not breath vapors.
4.2.2 Use a safety screen.
4.2.3 Use mechanical pipetting aides.
4.2.4 Do not heat above 90°C: EXPLOSION may result.
4.2.5 Avoid grinding surfaces, and avoid the use of ground-glass joints, sleeve bearings, and
glass stirrers: EXPLOSION may result.
4.2.6 Do not store near alkali metals: EXPLOSION may result.
4.2.7 Solutions of diazomethane decompose rapidly in the presence of solid materials such as
copper powder, calcium chloride, and boiling chips.
5. APPARA TUS AND MA TERIALS
5.1 Sampling equipment, for discrete or composite sampling.
5.1.1 Grab-sample bottle: Amber borosilicate or flint glass, 1-L or I-quart volume, fitted with
screw-caps lined with TFE-fluorocarbon. Aluminum foil may be substituted for TFE if
the sample is not corrosive. If amber bottles are not available, protect samples from
light. The container and cap liner must be washed, rinsed with acetone or methylene
chloride, and dried before use to minimize contamination.
5.1.2 Automatic sampler (optional): Must incorporate glass sample containers for the
collection of a minimum of 250 mL. Sample containers must be kept refrigerated at 4°C
and protected from light during compositing. If the sampler uses a peristaltic pump, a
minimum length of compressible silicone rubber tubing may be used. Before use,
however, the compressible tubing must be thoroughly rinsed with methanol, followed by
repeated rinsings with reagent water to minimize the potential for contamination of the
sample. An integrating flow meter is required to collect flow-proportional composites.
5.2 Glassware. (All specifications are suggested. Catalog numbers are included for illustration only.)
5.2.1 Separatory funnels: 60-mL and 2000-mL, with TFE-fluorocarbon stopcocks, ground-
glass or TFE stoppers.
5.2.2 Concentrator tube, Kuderna-Danish: 10-mL, graduated (Kontes K-570050-1025 or
equivalent). Calibration must be checked at the volumes employed in the test. Ground-
glass stopper is used to prevent evaporation of extracts.
101
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Method 675
5.2.3 Evaporative flask, Kuderna-Danish: 500-mL (Kontes K-570001-0500 or equivalent).
Attach to concentrator tube with springs.
5.2.4 Snyder column, Kuderna-Danish: Three-ball macro (Kontes K-503000-0121 or
equivalent).
5.2.5 Snyder column, Kuderna-Danish: Two-ball micro (Kontes K-569001-0219 or
equivalent).
5.2.6 Erlenmeyer flask: Pyrex, 250-mL with 24/40 ground-glass joint.
5.2.7 Vials: Amber glass, 10- to 15-mL capacity with TFE-fluorocarbon-lined screw-cap.
5.3 Boiling chips: Approximately 10/40 mesh. Heat at 400°C for 30 minutes or perform a Soxhiet
extraction with methylene chloride.
5.4 Water bath: Heated, with concentric ring cover, capable of temperature control (± 2°C). The
bath should be used in a hood.
5.5 Balance: Analytical, capable of accurately weighing to the nearest 0.0001 g.
5.6 Diazomethane generator: assemble from two test tubes 150mm long by 20mm ID, two Neoprene
rubber stoppers, and a source of nitrogen. The generator assembly is shown in Figure 1.
5.7 Glass wool: Acid-washed (Supelco 2-0383 or equivalent).
5.8 Gas chromatograph: Analytical system complete with gas chromatograph suitable for on-column
injection and all required accessories including syringes, analytical columns, gases, detector, and
strip-chart recorder. A data system is recommended for measuring peak areas.
5.8.1 Column 1: 180 cm long by 4mm ID glass, packed with 1.5% SP-225011.95% SP-2401
on Supelcoport (100/120 mesh) or equivalent. This column was used to develop the
method performance statements in Section 16. Alternative columns may be used in
accordance with the provisions described in Section 13.1.
5.8.2 Column 2: 180 cm long by 4 mm ID glass, packed with 5% OV-210 on Gas Chrom Q
(100/120 mesh) or equivalent.
5.8.3 Column 3: 180 cm long by 2 mm ID glass, packed with 0.1% SP-1000 on Carbopak C
(80/100 mesh) or equivalent.
5.8.4 Detector: Electron capture. This detector has proven effective in the analysis of
wastewaters for the parameters listed in the scope and was used to develop the method
performance statements in Section 15. Alternative detectors, including a mass
spectrometer, may be used n accordance with the provisions described in Section 13.1.
6. REAGENTS
6.1 Reagent water: Reagent water is defined as a water in which an interferent is not observed at the
method detection limit of each parameter of interest.
6.2 Acetone, hexane, methanol: Pesticide-quality or equivalent
6.3 Ethyl ether: Nanograde, redistilled in glass if necessary. Must be free of peroxides as indicated
by EM Quant test strips (available from Scientific Products Co., Cat. No. P1126-8, and other
suppliers). Procedures recommended for removal of peroxides are provided with the test strips.
After cleanup, 20 mL ethyl alcohol preservative must be added to each liter of ether.
102
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Method 615
6.4 Sodium sulfate: ACS, granular, acidified, anhydrous. Condition heating in a shallow tray at
400°C for a minimum of 4 hours to remove phthalates and other interfering organic substances.
Alternatively, heat 16 hours at 450 to 500°C in a shallow tray or perform a Soxhiet extraction
with methylene chloride for 48 hours. Acidify by slurrying 100 g sodium sulfate with enough
ethyl ether to just cover the solid. Add 0.1 mL concentrated sulfuric acid and mix thoroughly.
Remove the ether under vacuum. Mix 1 g of the resulting solid with 5 mL of reagent water and
measure the pH of the mixture. It must be below pH 4. Store at 130°C.
6.5 Hydrochloric acid (1+9): Add one volume of concentrated acid (ACS) to 9 volumes reagent
water.
6.6 Potassium hydroxide solution: 37% aqueous solution (W:V). Dissolve 37 g ACS-grade
potassium hydroxide pellets in reagent water and dilute to 100 mL.
6.7 Sulfuric acid solution (1+1): Slowly add 50 mL H 2 S0 4 (sp. gr. 1.84) to 50 mL of reagent water.
6.8 Sulfuric acid solution (1+3): Slowly add 25 mL H 2 S0 4 (sp. gr. 1.84) to 75 mL of reagent water.
Maintain at 4°C.
6.9 Carbitol: Diethylene glycol monoethyl ether, ACS. Available from Aldrich Chemical Co.
6.10 Diazald: N-methyl-N-nitroso-p-toluenesulfonamide, ACS. Available from Aldrich Chemical Co.
6.11 Silicic acid: Chromatographic grade, nominal 100 mesh. Store at 130°C.
6.12 Stock standard solutions (1.00 igI iL): Stock standard solutions can be prepared from pure
standard materials or purchased as certified solutions.
6.12.1 Prepare stock standard solutions by accurately weighing about 0.0100 g of pure acids.
Dissolve the material in pesticide-quality ethyl ether and dilute to volume in a 10-mL
volumetric flask. Larger volumes can be used at the convenience of the analyst. If
compound purity is certified at 96% or greater, the weight can be used without correction
to calculate the concentration of the stock standard. Commercially prepared stock
standards can be used at any concentration if they are certified by the manufacturer or
by an independent source.
6.12.2 Transfer the stock standard solutions into PTFE-sealed screw-cap vials. Store at 4°C and
protect from light. Stock standard solutions should be checked frequently for signs of
degradation or evaporation, especially just prior to preparing calibration standards from
them.
6.12.3 Stock standard solutions must be replaced after 1 week, or sooner if comparison with
check standards indicates a problem.
7. CAUBRA TION
7.1 Establish gas chromatographic operating parameters equivalent to those indicated in Table 1. The
gas chromatographic system must be calibrated using the external standard technique.
7.2 External standard calibration procedure:
7.2.1 For each parameter of interest, prepare working standards of the free acids at a minimum
of three concentration levels by adding accurately measured volumes of one or more
stock standards to a 10-mL volumetric flask containing 1.0 mL methanol and diluting to
volume with ethyl ether. One of the external standards should be representative of a
103
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Method 675
concentration near, but above, the method detection limit. The other concentrations
should correspond to the range of concentrations expected in the sample concentrates or
should define the working range of the detector.
7.2.2 Prepare calibration standards by esterification of 1.0O-mL volumes of the working
standards as described in Section 11. Using injections of 2 to 5 &L of each calibration
standard, tabulate peak height or area responses against the mass of free acid represented
by the injection. The results can be used to prepare a calibration curve for each para-
meter. Alternatively, the ratio of the response to the mass injected, defined as the
calibration factor (CF), can be calculated for each parameter at each standard concentra-
tion. If the relative standard deviation of the calibration factor is less than 10% over the
working range, the average calibration factor can be used in place of a calibration curve.
7.2.3 The working calibration curve or calibration factor must be verified on each working
shift by the preparation of one or more calibration standards. If the response for any
parameter varies from the predicted response by more than ± 10%, the test must be
repeated using a fresh calibration standard. Alternatively, a new calibration curve or
calibration factor must be prepared for that parameter.
7.3 Before using any cleanup procedure, the analyst must process a series of calibration standards
through the procedure to validate elution patterns and the absence of interference from the
reagents.
8. QuAliTY CONTROL
8.1 Each laboratory using this method is required to operate a formal quality control program. The
minimum requirements of this program consist of an initial demonstration of laboratory capability
and the analysis of spiked samples as a continuing check on performance. The laboratory is
required to maintain performance records to define the quality of data that is generated.
8.1.1 Before performing any analyses, the analyst must demonstrate the ability to generate
acceptable accuracy and precision with this method. This ability is established as
described in Section 8.2.
8.1.2 In recognition of the rapid advances occurring in chromatography, the analyst is
permitted certain options to improve the separations or lower the cost of measurements.
Eich time such modifications to the method are made, the analyst is required to repeat
the procedure in Section 8.2.
8.1.3 The laboratory must spike and analyze a minimum of 10% of all samples to monitor
continuing laboratory performance. This procedure is described in Section 8.4.
8.2 To establish the ability to generate acceptable accuracy and precision, the analyst must perform
the following operations.
8.2.1 Select a representative spike concentration for each compound (acid or ester) to be
measured. Using stock standards, prepare a quality control check sample concentrate in
acetone, 1000 times more concentrated than the selected concentrations.
8.2.2 Using a pipette, add 1.00 mL of the check sample concentrate to each of a minimum of
four 1000-mL aliquots of reagent water. A representative wastewater may be used in
place of the reagent water, but one or more additional aliquots must be analyzed to
104
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Method 615
determine background levels, and the spike level must exceed twice the background level
for the test to be valid. Analyze the aliquots according to the method beginning in
Section 10.
8.2.3 Calculate the average percent recovery (R), and the standard deviation of the percent
recovery (s), for the results. Wastewater background corrections must be made before
R and s calculations are performed.
8.2.4 Using the appropriate data from Table 2, determine the recovery and single operator
precision expected for the method, and compare these results to the values calculated in
Section 8.2.3. If the data are not comparable, review potential problem areas and repeat
the test.
8.3 The analyst must calculate method performance criteria and define the performance of the
laboratory for each spike concentration and parameter being measured.
8.3.1 Calculate upper and lower control limits for method performance as follows:
Upper Control Limit (UCL) = R + 3s
Lower Control Limit (LCL) = R - 3s
where R and s are calculated as in Section 8.2.3. The UCL and LCL can be used to
construct control charts 6 that are useful in observing trends in performance.
8.3.2 The laboratory must develop and maintain separate accuracy statements of laboratory
performance for wastewater samples. An accuracy statement for the method is defined
as R ± s. The accuracy statement should be developed by the analysis of four aliquots
of wastewater as described in Section 8.2.2, followed by the calculation of R and s.
Alternatively, the analyst may use four wastewater data points gathered through the
requirement for continuing quality control in Section 8.4. The accuracy statements
should be updated regularly. 6
8.4 The laboratory is required to collect in duplicate a portion of their samples to monitor spike
recoveries. The frequency of spiked sample analysis must be at least 10% of all samples or one
spiked sample per month, whichever is greater. One aliquot of the sample must be spiked and
analyzed as described in Section 8.2. If the recovery for a particular parameter does not fall
within the control limits for method performance, the results reported for that parameter in all
samples processed as part of the same set must be qualified as described in Section 14.3. The
laboratory should monitor the frequency of data so qualified to ensure that it remains at or
below 5%.
8.5 Before processing any samples, the analyst must demonstrate through the analysis of a l-L aliquot
of reagent water that all glassware and reagent interferences are under control. Each time a set
of samples is extracted or there is a change in reagents, a laboratory reagent blank must be
processed as a safeguard against laboratory contamination.
8.6 It is recommended that the laboratory adopt additional quality assurance practices for use with this
method. The specific practices that are most productive depend upon the needs of the laboratory
and the nature of the samples. Field duplicates may be analyzed to monitor the precision of the
sampling technique. When doubt exists over the identification of a peak on the chromatogram,
confirmatory techniques such as gas chromatography with a dissimilar column, specific element
105
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Method 615
detector, or mass spectrometer must be used. Whenever possible, the laboratory should perform
analysis of quality control materials and participate in relevant performance evaluation studies.
9. SAMPLE CouEcrIoN, PRESERVATiON, AND HANDLING
9.1 Grab samples must be collected in glass containers. Conventional sampling practices 7 should be
followed; however, the bottle must not be prerinsed with sample before collection. Composite
samples should be collected in refrigerated glass containers in accordance with the requirements
of the program. Automatic sampling equipment must be as free as possible of plastic and other
potential sources of contamination.
9.2 The samples must be iced or refrigerated at 4°C from the time of collection until extraction.
9.3 All samples must be extracted within 7 days and completely analyzed within 44) days of extraction.
10. SAMPLE EXTRACTiON
10.1 Mark the water meniscus on the side of the sample bottle for later determination of sample
volume. Pour the entire sample into a 2-L separatory funnel. Check the pH with wide-range pH
paper and adjust to pH less than 2 with sulfuric acid (1 + 1).
10.2 Add 150 niL ethyl ether to the sample bottle, cap the bottle, and shake 30 seconds to rinse the
walls. Transfer the solvent to the separatory funnel and extract the sample by shaking the funnel
for 2 minutes with periodic venting to release excess pressure. Allow the organic layer to separate
from the water phase for a minimum of 10 minutes. If the emulsion interface between layers is
more than one-third the volume of the solvent layer, the analyst must employ mechanical
techniques to complete the phase separation. The optimum technique depends upon the sample,
but may include stirring, filtration of the emulsion through glass wool, centrifugation, or other
physical means. Drain the aqueous phase into a 1000-mL Erlenmeyer flask and collect the extract
in a 250-mL ground-glass Erlenmeyer flask containing 2 mL of 37% potassium hydroxide
solution. Approximately 80 mL of the ethyl ether will remain dissolved in the aqueous phase.
10.3 Add a 50-mL volume of ethyl ether to the sample bottle and repeat the extraction a second time,
combining the extracts in the Erlenmeyer flask. Perform a third extraction in the same manner.
10.4 Add 15 mL reagent water and one or two clean boiling chips to the 250-mL flask and attach a
three-ball Snyder column. Prewet the Snyder column by adding 1 mL ethyl ether to the top.
Place the apparatus on a hot water bath (60 to 65°C), such that the bottom of the flask is bathed
in the water vapor. Although the ethyl ether will evaporate in about 15 minutes, continue heating
for a total of 60 minutes, beginning from the time the flask is placed on the water bath. Remove
the apparatus and let stand at room temperature for at least 10 minutes.
10.5 Transfer the solution to a 60-mL separatory funnel using 5 to 10 mL of reagent water. Wash the
basic solution twice by shaking for 1 minute with 20-mL portions of ethyl ether. Discard the
organic phase. The free acids remain in the aqueous phase.
10.6 Acidify the contents of the separatory funnel to pH 2 by adding 2 mL of cold (4°C) sulfuric acid
(1+3). Test with pH indicator paper. Add 20 mL ethyl ether and shake vigorously for 2
minutes. Drain the aqueous layer into the 250-mL Erlenmeyer, then pour the organic layer into
a 125-mL Erlenmeyer flask containing about 0.5 g of acidified anhydrous sodium sulfate. Repeat
106
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Method 615
the extraction twice more with 1O-mL aliquots of ethyl ether, combining all solvent in the 125-mL
flask. Allow the extract to remain in contact with the sodium sulfate for approximately 2 hours.
10.7 Assemble a Kuderna-Danish (K-D) concentrator by attaching a 10-mL concentrator tube to a
500-niL evaporative flask. Other concentration devices or techniques may be used in place of the
K-D if the requirements of Section 8.2 are met.
10.8 Pour the combined extract through a funnel plugged with acid-washed glass wool, and collect the
extract in the K-D in concentrator. Use a glass rod to crush any caked sodium sulfate during the
transfer. Rinse the Erlenmeyer flask and column with 20 to 30 mL of ethyl ether to complete the
quantitative transfer.
10.9 Add one to two clean boiling chips to the evaporative flask and attach a three-ball Snyder column.
Prewet the Snyder column by adding about 1 mL ethyl ether to the top. Place the K-D apparatus
on a hot water bath, 60 to 65°C, so that the concentrator tube is partially immersed in the hot
water, and the entire lower rounded surface of the flask is bathed with hot vapor. Adjust the
vertical position of the apparatus and the water temperature as required to complete the
concentration in 15 to 20 minutes. At the proper rate of distillation the balls of the column will
actively chatter but the chambers will not flood. When the apparent volume of liquid reaches
I mL, remove the K-D apparatus and allow it to drain and cool for at least 10 minutes.
10.10 Remove the Snyder column and rinse the flask and its lower joint into the concentrator tube with
1 to 2 mL of ethyl ether. A 5-mL syringe is recommended for this operation. Add a fresh
boiling chip. Attach a micro-Snyder column to the concentrator tube and prewet the column by
adding about 0.5 mL of ethyl ether to the top. Place the micro K-D apparatus on the water bath
so that the concentrator tube is partially immersed in the hot water. Adjust the vertical position
of the apparatus and the water temperature as required to complete concentration in 5 to 10
minutes. When the apparent volume of liquid reaches 0.5 mL, remove the micro K-D from the
bath and allow it to drain and cool. Remove the micro Snyder column and add 0.1 mL of
methanol. Rinse the walls of the concentrator tube while adjusting the volume to 1.0 mL with
ethyl ether.
11. EsTERIFIcA T1ON OF AciDs
11.1 Assemble the diazomethane generator (See Figure 1) in a hood using two test tubes 150 mm long
by 20 mm ID. Use neoprene rubber stoppers with holes drilled in them to accommodate glass
delivery tubes. The exit tube must be drawn to a point to bubble diazomethane through the
sample extract.
11.2 Add5mLofethylethertotheflrsttesttube. Add! mLofethylether, 1 mLofcarbitol, 1.5 mL
of 37% aqueous KOH, and 0.! to 0.2 g Diazald to the second test tube. Immediately place the
exit tube into the concentrator tube containing the sample extract. Apply nitrogen flow
(10 mL/min) to bubble diazomethane through the extract for 10 minutes or until the yellow color
of diazomethane persists.
11.3 Remove the concentrator tube and seal it with a neoprene or PTFE stopper. Store at room
temperature in a hood for 20 minutes.
11.4 Destroy any unreacted diazomethane by adding 0.1 to 0.2 g silicic acid to the concentrator tube.
Allow to stand until the evolution of nitrogen gas has stopped. Adjust the sample volume to
10.0 mL with hexane. Stopper the concentrator tube and store refrigerated if further processing
107
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Method 615
will not be performed immediately. It is recommended that the methylated extracts be analyzed
immediately to minimize any transesterification and other potential reactions that may occur.
Analyze by gas chromatography.
11.5 Determine the original sample volume by refilling the sample bottle to the mark and transferring
the water to a 1000-mL graduated cylinder. Record the sample volume to the nearest 5 mL.
12. CLEANUP AND SEPARA T1ON
12.1 No cleanup procedures were required to analyze the wastewaters described in Section 16. If
particular circumstances demand the use of a cleanup procedure, the analyst must determine the
elution profile and demonstrate that the recovery of each compound of interest for the cleanup
procedure is no less than 85%.
13. GAs CHROMATOGRAPHY
13.1 Table I summarizes the recommended operating conditions for the gas chromatograph. Included
in this table are estimated retention times and method detection limits that can be achieved by this
method. Examples of the separations achieved for the methyl esters are shown in Figures 2 to 3.
Other packed columns, chromatographic conditions, or detectors may be used if the requirements
of Section 8.2 are met. Capillary (open-tubular) columns may also be used if the relative standard
deviations of responses for replicate injections are demonstrated to be less than 6% and the
requirements of Section 8.2 are met.
13.2 Calibrate the system daily as described in Section 7.
13.3 Inject 1 to 5 L of the sample extract using the solvent-flush technique. 8 Record the volume
injected to the nearest 0.05 gEL, and the resulting peak size in area or peak height units. An
automated system that consistently injects a constant volume of extract may also be used.
13.4 The width of the retention-time window used to make identifications should be based upon
measurements of actual retention-time variations of standards over the course of a day. Three
times the standard deviation of a retention time can be used to calculate a suggested window size
for a compound. However, the experience of the analyst should weigh heavily in the
interpretation of chromatograms.
13.5 If the response for the peak exceeds the working range of the system, dilute the extract and
reanalyze.
13.6 If the measurement of the peak response is prevented by the presence of interferences, further
cleanup is required.
14. CALCULATiONS
14.1 Determine the concentration of individual compounds in the sample. Calculate the amount of free
acid injected from the peak response using the calibration curve or calibration factor in
Section 7.2.2. The concentration in the sample can be calculated as follows:
108
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Method 615
Equation 1
( A)(V )
Concentration, ,zgIL = ______
(V,)(V,)
where
A = Amount of material injected, in ng
V 1 = Voiwne of extract injected, in ,d.
V 1 = Volume of total extract, in 1 zL
V = Volume of water extracted, in inL
14.2 Report results in micrograms per liter as acid equivalent without correction for recovery data.
When duplicate and spiked samples are analyzed, report all data obtained with the sample results.
14.3 For samples processed as part of a set where the laboratory spiked sample recovery falls outside
of the control limits in Section 8.3, data for the affected parameters must be labeled as suspect.
15. GC/MS CONFIRMA T1ON
15.1 It is recommended that GCIMS techniques be judiciously employed to support qualitative
compound identifications made with this method. The mass spectrometer should be capable of
scanning the mass range from 35 amu to a mass 50 amu above the molecular weight of the methyl
ester of the acid herbicide. The instrument must be capable of scanning the mass range at a rate
to produce at least 5 scans per peak but not to exceed 7 seconds per scan utilizing a 70 V
(nominal) electron energy in the electron impact ionization mode. A GC to MS interface
constructed of all glass or glass-lined materials is recommended. A computer system should be
interfaced to the mass spectrometer that allows the continuous acquisition and storage on machine-
readable media of all mass spectra obtained throughout the duration of the chromatographic
program.
15.2 Gas chromatographic columns and conditions should be selected for optimum separation and
performance. The conditions selected must be compatible with standard GCIMS operating
practices. Chromatographic tailing factors of less than 5.0 must be achieved. 9
15.3 At the beginning of each day that confirmatory analyses are to be performed, the GCIMS system
must be checked to see that all decafluorotriphenyl phosphine (DFTPP) performance criteria are
achieved. ’ 0
15.4 To confirm an identification of a compound, the background-corrected mass spectrum of the
methyl ester must be obtained from the sample extract and compared with a mass spectrum from
a stock or calibration standard analyzed under the same chromatographic conditions. It is
recommended that at least 25 ng of material be injected into the GC/MS. The criteria below must
be met for qualitative confirmation.
15.4.1 All ions that are present above 10% relative abundance in the mass spectrum of the
standard must be present in the mass spectrum of the sample with agreement to ±10%.
For example, if the relative abundance of an ion is 30% in the mass spectrum of the
standard, the allowable limits for the relative abundanee of that ion in the mass spectrum
for the sample would be 20% to 40%.
109
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Method 815
15.4.2 The retention time of the compound in the sample must be within 6 seconds of the same
compound in the standard solution.
15.4.3 Compounds that have very similar mass spectra can be explicitly identified by GC/MS
only on the basis of retention time data.
15.5 Where available, chemical ionization mass spectra may be employed to aid in the qualitative
identification process.
15.6 Should these MS procedures fail to provide satisfactory results, additional steps may be taken
before reanalysis. These may include the use of alternate packed or capillary GC columns or
additional cleanup.
16. METHoD PERFORMANCE
16.1 The method detection limit (MDL) is defined as the minimum concentration of a substance that
can be measured and reported with 99% confidence that the value is above zero.U The MDL
concentrations listed in Table 1 were obtained from reagent water with an electron capture
detector. 1
16.2 in a single laboratory (West Coast Technical Services, Inc.), using reagent water and effluents
from publicly owned treatment works (POTW), the average recoveries presented in Table 2 were
obtained.’ The standard deviations of the percent recoveries of these measurements are also
included in Table 2.
110
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Method 615
References
1. “Pesticide Methods Evaluation,” Letter Report #33 for EPA Contract No. 68-03-2697. Available
from U.S. Enviromnental Protection Agency, Environmental Monitoring and Support Laboratory,
Cincinnati, Ohio.
2. ASTM Annual Book of Standards, Part 31, D3694, “Standard Practice for Preparation of Sample
Containers and for Preservation, “American Society for Testing and Materials, Philadelphia, PA,
p. 679, 1980.
3. “Carcinogens - Working with Carcinogens,” Department of Health, Education, and Welfare,
Public Health Service, Center for Disease Control, National Institute for Occupational Safety and
Health, Publication No. 77-206, Aug. 1977.
4. “OSHA Safety and Health Standards, General Industry,” (29 CFR 1910), Occupational Safety and
Health Administration, OSHA 2206, (Revised, January 1976).
5. “Safety in Academic Chemistry Laboratories,” American Chemical Society Publication,
Committee on Chemical Safety, 3rd Edition, 1979.
6. “Handbook for Analytical Quality Control in Water and Wastewater Laboratories,”
EPA-600/4-79-019, U. S. Environmental Protection Agency, Environmental Monitoring and
Support Laboratory - Cincinnati, Ohio 45268, March 1979.
7. ASTM Annual Book of Standards, Part 31, D3370, “Standard Practice for Sampling Water,”
American Society for Testing and Materials, Philadelphia, PA, p. 76, 1980.
8. Burke, J. A., “Gas Chromatography for Pesticide Residue Analysis; Some Practical Aspects,”
Journal of the Association of Official Analytical Chemists, 48, 1037 (1965).
9. McNair, H.M. and Bonelli, E. J., “Basic Chromatography,” Consolidated Printing, Berkeley,
California, p. 52, 1969.
10. Eichelberger, J.W., Harris, L.E., and Budde, W.L. “Reference Compound to Calibrate Ion
Abundance Measurement in Gas Chromatography-Mass Spectrometry,” Analytical Chemistry, 47,
995 (1975).
11. Glaser, J.A. et.al, “Trace Analysis for Wastewaters,” Environmental Science & Technology, 15,
1426 (1981).
111
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Method 615
Table 1. Chromatographic Conditions and Method Detection Limits
Retention Time Method
Detection
Parameter
(as methyl ester) Column I Column 2 Column 3 Limit pg/L
Dicamba 1.2 1.0 0.27
2,4-D 2.0 1.6 1.20
2,4,5-TP 2.7 2.0 0.17
2,4,5-T 3.4 2.4 0.20
2,4-DB 4.1 — 0.91
Dalapon 5.0 5.80
MCPP 3.4 192.00
MCPA 4.1 249.00
Dichlorprop 4.8 0.65
Dinoseb 11.2 0.07
Column 1 conditions: Supelcoport (100/120 mesh) coated with 1.5% SP-2250/ 1.95% SP-2401 packed
in a 1.8 m long by 4 mm ID glass column with 95% argon/5% methane carrier gas at a flow rate of
70 mljmin. Column temperature: isothermal at 185°C, except for MCPP, MCPA, dichlorprop and
dinoseb, where the column temperature was held at 140°C for 6 minutes and then programmed to 200°C
at 10°/mm. An electron capture detector was used to measure MDL.
Colwnn 2 conditions: Gas Chrom Q (100/120 mesh) coated with 5% OV-210 packed in a 1.8 m long
by 4 mmID glass column with 95% argon/5% methane carrier gas at a flow rate of 70 mL/min. Column
temperature: isothermal at 185°C.
Column 3 conditions: Carbopak C (80/100 mesh) coated with 0.1% SP-1000 packed in a 1.8 m long by
2 mm ID glass column with nitrogen carrier gas at a flow rate of 25 mL/min. Column temperature:
programmed at injection from 100°C to 150°C at 10°/mm.
772
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Method 615
Table 2. Single-Operator Accuracy and Precision*
Mean Standard
Sample Spike Recovery Deviation
Parameter Type (pg/U (96) (96)
2,4-D DW 10.9 75 4
MW 10.1 77 4
MW 200.0 65 5
Dalapon DW 23.4 66 8
MW 23.4 96 13
MW 468.0 81 9
2,4-DB DW 10.3 93 3
MW 10.4 93 3
MW 208.0 77 6
Dicamba DW 1.2 79 7
MW 1.1 86 9
MW 22.2 82 6
Dichiorprop DW 10.7 97 2
MW 10.7 72 3
MW 213.0 100 2
Dinoseb MW 0.5 86 4
MW 102.0 81 3
MCPA DW 2020.0 98 4
MW 2020.0 73 3
MW 214.00.0 97 2
MCPP DW 2080.0 94 4
MW 2100.0 97 3
MW 20440.0 95 2
2,4,5-T DW 1.1 85 6
MW 1.3 83 4
MW 25.5 78 5
2,4,5-TP DW 1.0 88 5
MW 1.3 88 4
MW 25.0 72 5
*A1l results based upon seven replicate analyses.
DW = Reagent water
MW = Municipal water
113
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Metfr4 615
Figure 1. Diazomethane Generator.
Glass Tubing
/
Tubel Tube2
114
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Method 615
2,4-D
/
2,4 1 5-TP
/
I I I I
I I
6.0
8.0
10.0
Retention Time (minutes)
Figure 2. Gas Chromatogram of Methyl Esters of Chionnated Herbicides on
Column 1. For Conditions, See Table 1.
A52-O -16
2,4,5-T
/
/
0
I I
2.0
4.0
115
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Akffiod 615
limo )
Figure 3. Gas Chromatogram of Methyl Esters of Chlorinated Herbicides
on Column 1. For Conditions, See Table 1.
MCPA
I—
/
0 2.0 4.0 6.0 8.0 10.0 12.0 14.0
16.0
A52-002 .1$
116
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Method 616.
The Determination of Certain
Carbon-, Hydrogen-, and
Oxygen-Containing Pesticides in
Municpal and Industrial
Waste water
-------
Method 616
The Determination of Certain Carbon-, Hydrogen-, and
Oxygen-Containing Pesticides in Municpal and Industrial Wastewater
1. SCOPE AND APPLICA TION
1.1 This method covers the determination of certain carbon-, hydrogen-, and oxygen-containing
pesticides. The following parameters can be determined by this method:
Paiamete, CAS No.
Cycloprate 54460-46-7
Kinoprene 42588-37-4
Methoprene 40596-69-8
Resmethrin 10453-86-8
1.2 This is a gas chromatographic (GC) method applicable to the determination of the compounds
listed above in municipal and industrial discharges as provided under 40 CFR 136.1. Any
modification of this method beyond those expressly permitted shall be considered a major
modification subject to application and approval of alternative test procedures under 40 CFR 136.4
and 136.5.
1.3 The method detection limit (MDL, defined in Section 15) for each parameter is listed in Table 2.
The MDL for a specific wastewater may differ from those listed, depending upon the nature of
interferences in the sample matrix.
1.4 The sample extraction and concentration steps in this method are essentially the Same as certain
other 600-series methods. Thus, a single sample may be extracted to measure the compounds
included in the scope of the methods. When cleanup is required, the concentration levels must
be high enough to permit selecting aliquots, as necessary, in order to apply appropriate cleanup
procedures.
1.5 This method is restricted to use by or under the supervision of analysts experienced in the use of
gas chromatography and in the interpretation of gas chromatograms. Each analyst must
demonstrate the ability to generate acceptable results with this method using the procedure
described in Section 8.2.
1.6 When this method is used to analyze unfamiliar samples for any or all of the compounds above,
compound identifications should be supported by at least one additional qualitative technique.
This method describes analytical conditions for a second gas chromatographic column that can be
used to confirm measurements made with the primary column. Section 14 provides gas
chromatograph/mass spectrometer (GC/MS) criteria appropriate for the qualitative confirmation
of compound identifications.
119
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Method 616
2. SUMMARY OF METHOD
2.1 A measured volume of sample, approximately 1 L, is extracted with methylene chloride using a
separatory funnel. The methylene chloride extract is dried and concentrated to 1.0 mL. Gas
chromatographic conditions are described which permit the separation and measurement of the
compounds in the extract by flame ionization detector/gas chromatography (GCIFID).’
2.2 This method provides a silica gel column cleanup procedure to aid in the elimination of
interferences which may be encountered.
3. INTERFERENCES
3.1 Method interferences may be caused by contaminants in solvents, reagents, glassware, and other
sample-processing apparatus that lead to discrete artifacts or elevated baselines in gas
chromatograms. All reagents and apparatus must be routinely demonstrated to be free from
interferences under the conditions of the analysis by running laboratory reagent blanks as
described in Section 8.5.
3.1.1 Glassware must be scrupulously cleaned. Clean all glassware as soon as possible after
use by thoroughly rinsing with the last solvent used in it. Follow by washing with hot
water and detergent and thorough rinsing with tap and reagent water. Drain dry, and
heat in an oven or muffle furnace at 400°C for 4 hours. Do not heat volumetric ware.
Some thermally stable materials, such as PCBs, may not be eliminated by this treatment.
Thorough rinsing with acetone and pesticide-quality hexane may be substituted for the
heating. After drying and cooling, seal and store glassware in a clean environment to
prevent any accumulation of dust or other contaminants. Store inverted or capped with
aluminum foil.
3.1.2 The use of high-purity reagents and solvents helps to minimize interference problems.
Purification of solvents by distillation in all-glass systems may be required.
3.2 Matrix interferences may be caused by contaminants that are coextracted from the sample. The
extent of matrix interferences will vary considerably from source to source, depending upon the
nature and diversity of the industrial complex or municipality sampled. The cleanup procedure
in Section 11 can be used to overcome many of these interferences, but unique samples may
require additional cleanup approaches to achieve the MDL listed in Table 2.
4. SAFETY
4.1 The toxicity or carcinogenicity of each reagent used in this method has not been precisely
defined; however, each chemical compound should be treated as a potential health hazard. From
this viewpoint, exposure to these chemicals must be reduced to the lowest possible level by
whatever means available. The laboratory is responsible for maintaining a current awareness file
of OSHA regulations regarding the safe handling of the chemicals specified in this method. A
reference file of material data handling sheets should also be made available to all personnel
involved in the chemical analysis. Additional references to laboratory safety are available and
have been identified 5 for the information of the analyst.
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Method 616
5. APPARATUS AND MATERIALS
5.1 Sampling equipment, for discrete or composite sampling.
5.1 .1 Grab-sample bottle: Amber borosilicate or flint glass, 1-L or 1-quart volume, fitted with
screw-caps lined with PTFE. Foil may be substituted for PTFE if the sample is not
corrosive. If amber bottles are not available, protect samples from light. The container
and cap liner must be washed, rinsed with acetone or methylene chloride, and dried
before use to minimize contamination.
5.1.2 Automatic sampler (optional): Must incorporate glass sample containers for the
collection of a minimum of 250 mL. Sample containers must be kept refrigerated at 4°C
and protected from light during compositing. If the sampler uses a peristaltic pump, a
minimum length of compressible silicone rubber tubing may be used. Before use,
however, the compressible tubing should be thoroughly rinsed with methanol, followed
by repeated rinsings with distilled water to minimize the potential for contamination of
the sample. An integrating flow meter is required to collect flow-proportional
composites.
5.2 Glassware. (All specifications are suggested. Catalog numbers are included for illustration only.)
5.2.1 Separatory funnel: 2000-mL, with PTFE stopcock.
5.2.2 Drying column: Chromatographic column 400 mm long by 10 mm ID with coarse frit.
5.2.3 Chromatographic column: 400 mm long by 19 mm ID with 250-mi. reservoir at the top
and PTFE stopcock (Kontes K-420290 or equivalent).
5.2.4 Concentrator tube, Kuderna-Danish: l0-mL, graduated (Kontes K-570050-1025 or
equivalent). Calibration must be checked at the volumes employed in the test. A
ground-glass stopper is used to prevent evaporation of extracts.
5.2.5 Evaporative flask, Kuderna-Danish: 250 mL (Kontes K-570001-0250 or equivalent).
Attach to concentrator or tube with springs.
5.2.6 Snyder column, Kuderna-Danish: Three-ball macro (Kontes K-503000-0121 or
equivalent).
5.2.7 Snyder column, Kuderna-Danish: Two-ball micro (Kontes K-569001-0219 or
equivalent).
5.2.8 Vials: Amber glass, 10- to 15-mL capacity with PTFE-lined screw-cap.
5.2.9 Graduated cylinder: 1000-mL.
5.2.10 Erlenmeyer flask: 250-mL.
5.3 Boiling chips: Approximately 10/40 mesh carborundum. Heat at 400°C for 4 hours or perform
a Soxhlet extraction with methylene chloride.
5.4 Water bath: Heated, capable of temperature control (±2°C). The bath should be used in a hood.
5.5 Balance: Analytical, capable of accurately weighing to the nearest 0.0001 g.
5.6 Gas chromatograph: Analytical system complete with gas chromatograph suitable for on-column
injection and all required accessories including syringes, analytical columns, gases, detector, and
strip-chart recorder. A data system is recommended for measuring peak areas.
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Method 616
5.6.1 Column 1: 180 cm long by 2 mm ID glass, packed with 3% SP2250 on Supelcoport
(100/120 mesh) or equivalent. This column was used to develop the method performance
statements in Section 15. Alternative columns may be used in accordance with the
provisions described in Section 12.1.
5.6.2 Column 2: 180 cm long by 2 mm ID glass, packed with 10% OV-210 on Supelcoport
(100/120 mesh) or equivalent.
5.6.3 Detector: Flame ionization detector (FID). This detector has proven effective in the
analysis of wastewaters for the compounds listed in the scope and was used to develop
the method performance statements in Section 15.
6. REAGENTS
6.1 Reagent water: Reagent water is defined as a water in which an interferent is not observed at the
method detection limit of each parameter of interest.
6.2 Methylene chloride, methanol, petroleum ether, ethyl ether, methyl 1-butyl ether, distilled-in-glass
quality or equivalent. Ethyl ether must be free of peroxides as indicated by EK Quant Test Strips
(available from Scientific Products Co., Catalog No. P1126-8 and other suppliers). Procedures
recommended for removal of peroxides are provided with the test strips.
6.3 Sodium sulike: ACS, granular, anhydrous; heated in a muffle furnace at 400°C overnight.
64 Silica gd: Davison Grade 923 (100/120 mesh). Purchase activated. To prepare for use, place
in a wide-mouth jar and heat overnight at 120 to 130°C. Seal tightly with PTFE or aluminum-
foil-lined screw-cap and cool to room temperature.
6.5 Sodium phosphate: Monobasic, monohydrate.
6.6 Sodium phosphate: Dibasic.
6.7 Stock standard solutions (1.00 g/ L): Stock standard solutions can be prepared from pure
standard materials or purchased as certified solutions.
6.7.1 Prepare stock standard solutions by accurately weighing about 0.0100 g of pure material.
Dissolve the material in distilled-in-glass quality methyl t-butyl ether and dilute to volume
in a 10-mL volumetric flask. Larger volumes can be used at the convenience of the
analyst. If compound purity is certified at 96% or greater, the weight can be used
without correction to calculate the concentration of the stock standard. Commercially-
prepared stock standards can be used at any concentration if they are certified by the
manufacturer or by an independent source.
6.7.2 Transfer the stock standard solutions into PTFE-sealed screw-cap bottles. Store at 4°C
and protect from light. Frequently check standard solutions for signs of degradation or
evaporation, especially just prior to preparing calibration standards from them.
6.7.3 Stock standard solutions must be replaced after 6 months, or sooner if comparison with
check standards indicates a problem.
7. CALIBRATION
7.1 Establish gas chromatographic operating parameters equivalent to those indicated in Table 2. The
gas chromatographic system can be calibrated using the external standard technique (Section 7.2)
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Method 616
For each compound of interest, prepare calibration standards at a minimum of three
concentration levels by adding volumes of one or more stock standards to a volumetric
flask and diluting to volume with methyl t-butyl ether. One of the external standards
should be at a concentration near, but above, the method detection limit. The other
concentrations should correspond to the range of concentrations expected in the sample
concentrations or should define the working range of the detector.
7.2.2 Using injection of 1 to 5 L of each calibration standard, tabulate peak height or area
responses against the mass injected. The results can be used to prepare a calibration
curve for each compound. Alternatively, the ratio of the response to the mass injected,
defined as the calibration factor (CF), can be calculated for each compound at each
standard concentration. If the relative standard deviation of the calibration factor is less
than 10% over the working range, the average calibration factor can be used in place of
a calibration curve.
7.2.3 The working calibration curve or calibration factor must be verified on each working
shift by the measurement of one or more calibration standards. If the response for any
compound varies from the predicted response by more than 10%, the test must be
repeated using a fresh calibration standard. Alternatively, a new calibration curve or
calibration factor must be prepared for that compound.
7.3 Internal standard calibration procedure: To use this approach, the analyst must select one or more
internal standards similar in analytical behavior to the compounds of interest. The analyst must
further demonstrate that the measurement of the internal standard is not affected by method or
matrix interferences. Due to these limitations, no internal standard applicable to all samples can
be suggested.
7.3.1
Prepare calibration standards at a minimum of three concentration levels for each
compound of interest by adding volumes of one or more stock standards to a volumetric
flask. To each calibration standard, add a known constant amount of one or more
internal standards, and dilute to volume with methyl t-butyl ether. One of the
standards should be at a concentration near, but above, the method detection limit. The
other concentrations expected in the sample concentrates or should define the working
range of the detector.
7.3.2 Using injections of 1 to 5 1 &L of each calibration standard, tabulate the peak height or
area responses against the concentration for each compound and internal standard.
Calculate response factors (RF) for each compound as follows:
or the internal standard technique (Section 7.3).
7.2 External standard calibration procedure.
7.2.1
123
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Method 676
Equation 1
= ( A,)(C,, )
(A 1 ,)(C,)
where
A, = Response for the parameter to be measured
4, = Response for the internal standard
C i = Concentration of the internal standard, in g/L
C, = Concentration of the parameter to be measured, in zgIL
7.3.3 If the RF value over the working range is constant, less than 10% relative standard
deviation, the RF can be assumed to be invariant and the average RF can be used for
calculations. Alternatively, the results can be used to plot a calibration curve or response
ratios, A/A against RF. The working calibration curve or RF must be verified on each
working shift by the measurement of one or more calibration standards. If the response
for any compound varies from the predicted response by more than 10%, the test must
be repeated using a fresh calibration standard. Alternatively, a new calibration curve
must be prepared for that compound.
7.4 Before using any cleanup procedure, the analyst must process a series of calibration standards
through the procedure to validate elution patterns and the absence of interferences from the
reagents.
8. QuAun’ CONTROL
8.1 Each laboratory using this method is required to operate a formal quality control program. The
minimum requirements of this program consist of an initial demonstration of laboratory capability
and the analysis of spiked samples as a continuing check on performance. The laboratory is
required to maintain performance records to define the quality of data that is generated.
8.1.1 Before performing any analyses, the analyst must demonstrate the ability to generate
acceptable accuracy and precision with this method. This ability is established as
described in Section 8.2.
8.1.2 In recognition of the rapid advances occurring in chromatography, the analyst is
permitted certain options to improve the separations or lower the cost of measurements.
Each time such modifications to the method are made, the analyst is required to repeat
the procedure in Section 8.2.
8.1.3 The laboratory must spike and analyze a minimum of 10% of all samples to monitor
continuing laboratory performance. This procedure is described in Section 8.4.
8.2 To establish the ability to generate acceptable accuracy and precision, the analyst must perform
the following operations.
8.2.1 Select a representative spike concentration for each compound to be measured. Using
stock standards, prepare a quality control check sample concentrate in methyl t-butyl
ether, 1000 times more concentrated than the selected concentrations.
124
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Method 616
8.2.2 Using a pipette, add 1.00 mL of the check sample concentrate to each of a minimum of
four l000-mL aliquots of reagent water. A representative wastewater may be used in
place of the reagent water, but one or more additional aliquots must be analyzed to
determine background levels, and the spike level must exceed twice the background level
for the test to be valid. Analyze the aliquots according to the method beginning in
Section 10.
8.2.3 Calculate the average percent recovery (R), and the standard deviation of the percent
recovery (s), for the results. Wastewater background corrections must be made before
R and s calculations are performed.
8.2.4 Using the appropriate data from Table 3, determine the recovery and single operator
precision expected for the method, and compare these results to the values measured in
Section 8.2.3. If the data are not comparable, the analyst must review potential problem
areas and repeat the test.
8.3 The analyst must calculate method performance criteria and define the performance of the
laboratory for each spike concentration and parameter being measured.
8.3.1 Calculate upper and lower control limits for method performance as follows:
Upper Control Limit (UCL): R + 3s
Lower Control Limit (LCL): R - 3s
where R and s are calculated as in Section 8.2.3. The UCL and LCL can be used to
construct control charts 6 that are useful in observing trends in performance.
8.3.2 The laboratory must develop and maintain separate accuracy statements of laboratory
performance for wastewater samples. An accuracy statement for the method is defined
as R ± s. The accuracy statement should be developed by the analysis of four aliquots
of wastewater as described in Section 8.2.2, followed by the calculation of R and s.
Alternatively, the analyst may use four wastewater data points gathered through the
requirement for continuing quality control in Section 8.4. The accuracy statements
should be updated regularly.
8.4 The laboratory is required to collect in duplicate a portion of their samples to monitor spike
recoveries. The frequency of spiked sample analysis must be at least 10% of all samples or one
sample per month, whichever is greater. One aliquot of the sample must be spiked and analyzed
as described in Section 8.2. If the recovery for a particular compound does not fall within the
control limits for method performance, the results reported for that compound in all samples
processed as part of the same set must be qualified as described in Section 13.3. The laboratory
should monitor the frequency of data so qualified to ensure that it remains at or below 5%.
8.5 Before processing any samples, the analyst should demonstrate through the analysis of a 1-L
aliquot of reagent water that all glassware and reagent interferences are under control. Each time
a set of samples is extracted or there is a change in reagents, a laboratory reagent blank should
be processed as a safeguard against laboratory contamination.
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Method 616
8.6 It is recommended that the laboratory adopt additional quality assurance practices for use with this
method. The specific practices that are most productive depend upon the needs of the laboratory
and the nature of the samples. Field duplicates may be analyzed to monitor the precision of the
sampling technique. When doubt exists over the identification of a peak on the chromatogram,
confirmatory techniques such as gas chromatography with a dissimilar column, specific element
detector, or mass spectrometer must be used. Whenever possible, the laboratory, should perform
analysis of standard reference materials and participate in relevant performance evaluation studies.
9. SAMPLES C0LLEc770N, PRESERVATION. AND HANDLING
9.1 Grab samples must be collected in glass containers. Conventional sampling practices’ should be
followed; however, the bottle must not be prerinsed with sample before collection. Composite
samples should be collected in refrigerated glass containers in accordance with the requirements
of the program. Automatic sampling equipment must be as free as possible of plastic and other
potential sources of contamination.
9.2 The samples must be iced or refrigerated at 4°C from the time of collection until extraction.
93 Adjust the pH of the sample to 6.8 by addition of 2 g each of monobasic and dibasic sodium
phosphate per liter of sample.
10. SAMPLE EXTRACTION
10.1 Mark the water meniscus on the side of the sample bottle for later determination of sample
volume. Pour the entire sample into a 2-L separatory funnel. Add 2 g each of monobasic sodium
phosphate and dibasic sodiuni phosphate to the sample to adjust the pH to 6.8.
10.2 Add 60 iiiL of methylene chloride to the sample bottle, seal, and shake 30 seconds to rinse the
inner walls. Transfer the solvent to the separatory funnel and extract the sampló by shaking the
funnel for 2 minutes with periodic venting to release excess pressure. Allow the organic layer to
separate from the water phase for a minimum of 10 minutes. If the emulsion interface between
layers is more than one-third the volume of the solvent layer, the analyst must employ mechanical
techniques to complete the phase separation. The optimum technique depends upon the sample,
but may include stirring, filtration of the emulsion through glass wool, centrifugation, or other
physical methods. Collect the methylene chloride extract in a 250-mL Erleiuneyer flask.
10.3 Add a second 60-mL volume of methylene chloride to the sample bottle and repeat the extraction
procedure a second time, combining the extracts in the Erlenmeyer flask. Perform a third
extraction in the same manner.
10.4 Assemble a Kuderna-DaIthh (K-D) concentrator by attaching a 10-mL concentrator tube to a 250-
mL evaporative flask. Other concentration devices or techniques may be used in place of the K-D
if the requirements of Section 8.2 are met.
10.5 Pour the combined extract through a drying colum containing about 10 cm of anhydrous sodium
sulfate, and collect the extract in the IC-D concentrator. Rinse the Erlenmeyer flask and column
with 20 to 30 mL of methylene chloride to complete the quantitative transfer. Once the flask rinse
has passed through the drying column, rinse the column with 30 to 40 mL of methylene chloride.
126
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Method 616
10.6 Add one or two clean boiling chips to the evaporative flask and attach a three-ball Snyder column.
Prewet the Snyder column by adding about 1 mL methylene chloride to the top. Place the K-D
apparatus on a hot water bath, 60 to 65°C, so that the concentrator tube Is partially immersed in
the hot water, and the entire lower rounded surface of the flask is bathed with hot vapor. Adjust
the vertical position of the apparatus and the water temperature as required to complete the
concentration in 15 to 20 minutes. At the proper rate of distillation, the balls of the column will
actively chatter but the chambers will not flood with condensed solvent. When the apparent
volume of liquid reaches 3 to 4 mL, remove the K-D apparatus and allow it to drain and cool for
at least 10 minutes.
10.7 Remove the macro-Snyder column and rinse the flask and its lower joint into the concentrator tube
with 1 to 2 mL of methylene chloride. Adjust the sample extract volume to 5 mL with methylene
chloride.
10.8 Stopper the concentrator tube and store refrigerated if further processing will not be performed
immediately. If the extract is to be stored longer than 2 days, transfer the extract to a screw-
capped vial with a PTFE-Iined cap. If the sample extract requires no further cleanup, proceed to
Section 10.9. If the sample requires cleanup, proceed to Section 11.
10.9 Add one or two boiling chips and attach a two-ball micro-Snyder column to the concentrator tube.
Prewet the micro-Snyder column with methylene chloride and concentrate the solvent extract to
1 mL as before.
10.10 Add 20 mL of methyl t-butyl ether to the concentrator tube and reconcentrate the solvent extract
as before. When an apparent volume of 0.5 mL is reached, or the solution stops boiling, remove
the K-D apparatus and allow it to drain and cool for 10 minutes.
10.11 Remove the micro-Sayder column and adjust the volume of the extract to 1.0 mL with methyl t-
butyl ether. Transfer the extract to an appropriate container for subsequent GC analysis.
10.12 Determine the original sample volume by refilling the sample bottle to the mark and transferring
the water to a 1000-mL graduated cylinder. Record the sample volume to the nearest 5 mL.
11. CLEANUP AND SEPARATION
11.1 Cleanup procedures may not be necessary for a relatively clean sample matrix. The cleanup
procedure recommended in this method has been used for the analysis of various clean waters and
industrial effluents. If particular circumstances demand the use of an alternative cleanup
procedure, the analyst must determine the elution profile and demonstrate that the recovery of
each compound of interest is no less than 85%.
11.2 The following silica gel column cleanup procedure has been demonstrated to be applicable to the
four C, H, and 0 pesticides listed in Table 1.
11.2.1 Deactivate silica gel by mixing 100 mL of acetone, 1.2 mL of distilled water, and 20 g
of silica gel thoroughly for 30 minutes in a 250-mL beaker. Transfer the slurry to a
chromatographic column (silica gel is retained with a plug of glass wool). Allow the
solvent to elute from the column until the silica gel is almost exposed to the air. Wash
the column sequentially with 10 mL of acetone, two 10-mL portions of methylene
chloride, and three 10-mL portions of petroleum ether. Use a column flow rate of 2 to
127
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Method 616
2.5 mL/min throughout the wash and elution profiles. Add an additional 50 mL of
petroleum ether to the head of the column.
11.2.2 Quantitatively add the methylene chloride extract from Section 10.8 to the head of the
column. Allow the solvent to elute from the column until the silica gel is almost exposed
to the air. Elute the column with 25 mL of petroleum ether. Discard this fraction.
11.2.3 Elute the column with 50 mL of 6% ethyl ether in petroleum ether (Fraction 1) and
collect eluate in a K-I) apparatus. Repeat process with 50 mL of 15% ethyl ether in
petroleum ether (Fraction 2), add 100 mL of 50% ethyl ether in petroleum ether
(Fraction 3). Collect each fraction in a separate K-I) apparatus. The elution patterns for
the C, H, and 0 pesticides are shown in Table 1. Concentrate each fraction to 1 mL as
described in Sections 10.9, 10.10, and 10.11. Proceed with gas chromatographic
analysis.
11.2.4 The above-mentioned fractions can be combined before concentration at the discretion of
the analyst.
12. G4s CHROMATOGRAPHY
12.1 Table 2 summarizes the recommended operating conditions for the gas chromatograph. Included
In this table are estimated retention times and method detection limits that can be achieved by this
method. Examples of the separations achieved by Columns 1 and 2 are shown in Figures 1 and 2.
Other packed columns, chromatographic conditions, or detectors may be used if the requirements
of Section 8.2 are met. Capillary (open-tubular) columns may also be used if the relative standard
deviations of responses for replicate injections are demonstrated to be less than 6% and the
requirements of Section 8.2 are met.
12.2 Calibrate the gas chromatograpliic system daily as described in Section 7.
12.3 If the Internal standard approach is being used, the analyst must not add the internal standard to
the s2mple extracts until immediately before injection into the instrument. Mix thoroughly.
12.4 Inject 1 to 5 pL of the sample extract using the solvent flush technique. Record the volume
injected to the nearest 0.05 pL, and the resulting peak sizes in area or peak height units. An
automated system that consistently injects a constant volume of extract may also be used.
12.5 Th. width of the retention-time window used to make identifications should be based upon
n easur n of actual retention-time variations of standards over the course of a day. Three
times the st nd rd deviation of a retention time for a compound can be used to calculate a
suggested window size; however, the experience of the analyst should weigh heavily in the
Interpretation of chromatograms.
12.6 If the response for the peak exceeds the working range of the system, dilute the extract and
reanalyze.
12.7 If the measurement of the peak response is prevented by the presence of interferences, further
cleanup is required.
128
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Method 616
13. CALCULATiONS
13.1 Determine the concentration of individual compounds in the sample.
13.1.1 If the external standard calibration procedure is used, calculate the amount of material
injected from the peak response using the calibration curve or calibration factor in
Section 7.2.2. The concentration in the sample can be calculated as follows:
Equation 2
Concentration, gIL = ( A)(V, )
(V )(V ,)
where
A = Amount of material infected, in ng
V 1 = Voiwne of extract injected, in p .L
V 1 = Volume of total extract, in uL
V , = Volume of water extracted, in mL
13.1.2 If the internal standard calibration procedure was used, calculate the concentration in the
sample using the response factor (RE) determined in Section 7.3.2 as follows:
Equation 3
Concentration, p g/L = ( ) (
(An) (RE) (“s)
where
A, = Response for parameter to be measured
4, = Response for the internal standard
I , = Amount of internal standard added to each extract, in ,zg
V 0 = Volume of water extracted, in L
13.2 Report results in micrograms per liter without correction for recovery data. When duplicate and
spiked samples are analyzed, report all data obtained with the sample results.
13.3 For samples processed as part of a set where the laboratory spiked sample recovery falls outside
of the control limits in Section 8.3, data for the affected compounds must be labeled as suspect.
14. GC/MS CONFFRMA TION
14.1 It is recommended that GCIMS techniques be judiciously employed to support qualitative
identifications made with this method. The mass spectrometer should be capable of scanning the
mass range from 35 amu to a mass 50 amu above the molecular weight of the compound. The
instrument must be capable of scanning the mass range at a rate to produce at least 5 scans per
peak but not to exceed 7 seconds per scan utilizing a 70 V (nominal) electron energy in the
electron impact ionization mode. A GC to MS interface constructed of all glass or glass-lined
129
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Method 616
materials is recommended. When using a fused silica capillary column, the column outlet should
be threaded through the interface to within a few millmeters of the entrance to the source
ionization chamber. A computer system should be interfaced to the mass spectrometer that allows
the continuous acquisition and storage on machine-readable media of all mass spectra obtained
throughout the duration of the chroinatographic program.
14.2 Gas chromatographic columns and conditions should be selected for optimum separation and
performance. The conditions selected must be compatible with standard GC/MS operating
practices. Chromatographic tailing factors of less than 5.0 must be achieved.’ 0
14.3 At the beginning of each day that confirmatory analyses are to be performed, the GCIMS system
must be checked to see that all DFTPP performance criteria are achieved. 9
14.4 To confirm an identification of a compound, the background-corrected mass spectrum of the
compound must be obtained from the sample extract and compared with a mass spectrum from
a stock or calibration standard analyzed under the same chromatographic conditions. It is
recommended that at least 25 ng of material be injected into the GCIMS. The criteria below must
be met for qualitative confirmation.
14.4.1 The molecular ion and all other ions that are present above 10% relative abundance in
the mass be present in the mass spectrum of the sample with agreement to ±10%. For
example, if the relative abundance of an ion is 30% in the mass spectrum of the standard,
the allowable limits for the relative abundance of that ion in the mass spectrum for the
sample would be 20 to 40%.
14.4.2 The retention time of the compound in the sample must be within 6 seconds of the same
compound in the standard solution.
14.4.3 Compounds that have very similar mass spectra can be explicitly identified by GCIMS
only on the basis of retention time data.
14.5 Where available, chemical ionization mass spectra may be employed to aid in the qualitative
identification process.
14.6 Should these MS procedures fail to provide satisfactory results, additional steps may be taken
before reanalysis. These may include the use of alternate packed or capillary GC columns or
additional cleanup (Section 11).
15. METHOD PERFORMANCE
15.1 The method detection limit (MDL) is defined as the minimum concentration of a substance that
can be measured and reported with 99% confidence that the value is above zero.” The MDL
concentrations listed in Table 2. Similar results were obtained using reagent water were achieved
using representative wastewaters.
15.2 This method has been tested for linearity of recovery from spiked reagent water and has been
demonstrated to be applicable over the concentration range 10 x MDL to 1000 x MDL.
15.3 In a single laboratory, Battelle Columbus Laboratories, using spiked wastewater samples, the
average recoveries presented in Table 3 were obtained after silica gel cleanup. Seven replicates
of each of two different wastewaters were spiked and analyzed. The standard deviation of the
percent recovery is also included in Table 3•1
130
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Method 616
References
1. “Development of Methods for Pesticides in Wastewater,” Report for EPA Contract 68-03-2956
(In preparation).
2. ASTM Annual Book of Standards, Part 31, D3694, “Standard Practice for Preparation of Sample
Containers and for Preservation,” American Society for Testing and Materials, Philadelphia, PA,
p. 679, 1980.
3. “Carcinogens: Working with Carcinogens,” Department of Health, Education, and Welfare,
Public Health Service, Center for Disease Control, National Institute for Occupational Safety and
Health, Publication No. 77-206, August, 1977.
4. “OSHA Safety and Health Standards, General Industry,” (29 CFR 1910), Occupational Safety and
Health Administration, OSHA 2206 (Revised, January 1976).
5. “Safety in Academic Chemistry Laboratories,” American Chemical Society Publication,
Committee on Chemical Safety, 3rd Edition, 1979.
6. “Handbook for Analytical Quality Control in Water and Wastewater Laboratories,” EPA-600/4-
79-019, U.S. Environmental Protection Agency, Environmental Monitoring and Support
Laboratory Cincinnati, Ohio, March 1979.
7. ASTM Aimual Book of Standards, Part 31, D3370, “Standard Practice for Sampling Water,”
American Society for Testing and Materials, Philadelphia, PA, p. 76, 1980.
8. Burke, J. A., ‘Gas Chromatography for Pesticide Residue Analysis; Some Practical Aspects,”
Journal of the Association of Official Analytical Chemists, 48, 1037 (1965).
9. Eichelberger, J. W., Harris, L. E., and Budde, W. L., “Reference Compound to Calibrate Ion
Abundance Measurement in Gas Chromatography: Mass Spectrometry,” Analytical Chemistry,
47, 995 (1975).
10. McNair, H.M., and Bonelli, E.J., “Basic Chromatography”, Consolidated Printing, Berkeley,
California, p. 52 (1969).
11. Glaser, J.A. et al., “Trace Analysis for Wastewaters”, Environmental Science and Technology,
15, 1426 (1981)
131
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Metl d 616
Table 1. Elution Characteristics of the C, H, And 0 Compounds from 6%
Deactivated Silica Gel
Recovery in Specified fraction (a)
Pa,ameter F l F2 F3 Total
Cycloprate 97 ND(a) ND 97
Kinoprene 100 ND ND 100
Methoprene ND 101 <1 101
Resmethrin 65 27 ND 92
(a) Elution solvents are 50 mL each of the following:
Fl =6% ethyl ether in petroleum ether
F2 = 15% ethyl ether in petroleum ether
F3 = 50% ethyl ether
(b) ND = Not dçtected
Table 2. Chromatographic Conditions and Method Detection Limits
Retention Time (Mis.) MDL
Parameter Column I Column 2 (pg/Li
Cycloprate 3.6 3.9 21
4.4 5.5 18
Methoprene 5.5 6.5 22
Resmethrin 8.4 8.9 36
Column I conditions: Supelcoport (100/120 mesh) coated with 3% SP-2250 packed in a 1.8 m long by
2 mmID glass column with helium carrier gas at a flow rate of 30 mL/min. Column temperature is
programmed from 180°C to 240°C at 8°C/mm, injector temperature is 280°C and detector is 300°C.
A flame ionization detector is used.
Column 2 conditions: Supeicoport (100/120 mesh) coated with 10% OV-210 packed in a 1.8 m long by
2 mm ID glass columns with helium carrier gas at a flow rate of 30 mlJmin. Column temperature is
programmed from 180°C to 240°C at 4°C/mm, injector temperature is 280°C and detector is 300°C.
A flame ionization detector is used.
132
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Method 616
Table 3. Single-Laboratory Accuracy and Precision(a)
Mean Standard
Sample Backgroud Spike Recovery Deviation Number of
Parameter Type(b) pg/L(c) pg/L % Replicates
Cycloprate 1 ND 100 84 14 7
1 ND 1000 94 4 7
Kinoprene 1 ND 100 89 6 7
1 ND 1000 92 6 7
Methoprene 1 ND 100 93 13 7
1 ND 1000 90 4 7
Resmethrin 1 ND 100 86 8 7
1 ND 1000 91 3 7
(a) Column 1 conditions were used.
(b) 1 = Columbus POTW secondary effluent
(c) ND = Not detected
133
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Method 616
Metho
Cyclopate Resmethrin
I I I I I I T T TTh —
0 38 4.8 5.8 6.8 7.8 8.8 9.8 10.8 11.8 12.8
Retention Tkn. (minutes)
M200211
Figure 1. GC-FID Chromatogram of 200 ng Each of C, H, and 0 Compounds
(Column 1).
134
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Method 616
Retention Time (minutes) A52-002 .18
Figure 2. GC-FID Chromatogram of 200 ng Each of C, H, and 0 Compounds
(Column 2).
Methoprene
Resmethrin
Kinoprene
N
3.8
4.8
8.8
9.8
10.8
135
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Method 617
The Determination of
Organohalide Pesticides and
PCBs in Municipal and Industrial
Waste water
-------
Method 617
The Determination of Organohalide Pesticides and PCBS in Municipal
and Industrial Wastewater
SCOPE AND APPLICA flON
1.1 This method covers the determination of certain organohalide pesticides and PCBs. The following
parameters can be determined by this method:
Parameter Storet No. CAS No.
Aidrin 39330 309-00-2
a-BHC 39337 319-84-6
8-BHC 39338 3 19-85-7
ô-BHC 39259 3 19-86-8
y-BHC 39340 58-89-9
Captan 39640 133-06-2
Carbophenothion 786-19-6
Chiordane 39350 5 103-74-2
4,4’-DDD 39310 72-54-8
4,4’-DDE 39320 72-55-9
4,4’-DDT 39300 50-29-3
Dichioran 99-30-9
Dicofol 39780 115-32-2
Die ldrin 39380 60-57-1
Endosulfan I 34356 959-98-8
Endosulfan II 34361 33213-65-9
Endosulfansulfate 34351 1031-07-8
Endrin 39390 72-20-8
Endrinaldehyde 34366 7421-93-4
Heptachior 39410 76-44-8
Heptachior epoxide 39420 1024-57-3
Isodrin 39430 465-73-6
139
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Method 617
Parameter Storet No. CAS No.
Methoxychlor 39480 72-43-5
Mirex 39755 2385-85-5
PCNB 39029 82-68-8
Perthane 39034 72-56-0
Strobane 8001-50-1
Toxaphene 39400 8001-35-2
Trifluralin 39030 1582-09-8
PCB-10 16 34671 12674-11-2
PCB-1221 39488 11104-28-2
PCB-1232 39492 11141-16-5
PCB-1242 39496 53469-21-9
PCB-1248 39500 12672-29-6
PCB-1254 39504 11097-69-1
PCB-1260 39508 11096-82-5
1.2 This is a gas chromatographiC (GC) method applicable to the determination of the compounds
listed above in industrial and municipal discharges as provided under 40 CFR 136.1. Any
modification of this method beyond those expressly permitted shall be considered a major
modification subject to application and approval of alternative test procedures under 40 CFR 136.4
and 136.5.
1.3 The method detection limit (MDL, defined in Section 15) for many of the parameters are listed
in Table 1. The MDL lbr a specific wastewater may differ from those listed, depending upon the
nature of interferences in the sample matrix.
1.4 The sample extraction and concentration steps in this method are essentially the same as in
Method 614. Thus, a single sample may be extracted to measure the parameters included in the
scope of both of these methods. When cleanup is required, the concentration leveLs must be high
enough to pennit selecting aliquots, as necessary, in order to apply appropriate cleanup
procedures. Under gas chromatography, the analyst is allowed the latitude to select
chromatographic conditions appropriate for the simultaneous measurement of combinations of
these parameters (see Section 12).
1.5 Thismethod isrestrictadtO useby or underthesupe isioflofana exPenenced in the useof
gas chromatography and in the interpretatIon of gas chromatograms. Each analyst must
demonstrate the ability to generate acceptable results with this method using the procedure
described in SectIon 8.2.
1.6 Whenthis method isusedtoanalyze unfamiliarsampleS foranyorall of the compounds above,
compound identifications should be supported by at least one additional qualitative technique.
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This method describes analytical conditions for a second gas chromatographic column that can be
used to confirm measurements made with the primary column. Section 14 provides gas
chromatograph/mass spectrometer (GC/MS) criteria appropriate for the qualitative confirmation
of compound identifications.
2. SUMMARY OF METHOD
2.1 A measured volume of sample, approximately 1 L, is exracted with 15% methylene chloride in
hexane using a separatory funnel. The extract is dried and concentrated to a volume of 10 mL
or less. Gas chromatographic conditions are described which permit the separation and
measurement of the compounds in the extract by electron capture gas chromatography.
2.2 Method 617 represents an editorial revision of two previously promulgated U.S. EPA methods
for pesticides and for PCBs.’ While complete method validation data is not presented herein, the
method has been in widespread use since its promulgation, and represents the state of the art for
the analysis of such materials.
2.3 This method provides selected cleanup procedures to aid in the elimination of interferences which
may be encountered.
3. INTERFERENCES
3.1 Method interferences may be caused by contaminants in solvents, reagents, glassware, and other
sample processing apparatus that lead to discrete artifacts or elevated baselines in gas
chromatograms. All reagents and apparatus must be routinely demonstrated to be free from
interferences under the conditions of the analysis by running laboratory reagent blanks as
described in Section 8.5.
3.1.1 Glassware must be scrupulously cleaned. 2 Clean all glassware as soon as possible after
use by thoroughly rinsing with the last solvent used in it. Follow by washing with hot
water and detergent and thorough rinsing with tap and reagent water. Drain dry, and heat
in an oven or muffle furnace at 400°C for 15 to 30 minutes. Do not heat volumetric
ware. Thermally stable materials, such as PCBs, may not be eliminated by this
treatment. Thorough rinsing with acetone and pesticide-quality hexane may be substituted
for the heating. After drying and cooling, seal and store glassware in a clean
environment to prevent any accumulation of dust or other contaminants. Store inverted
or capped with aluminum foil.
3.1.2 The use of high-purity reagents and solvents helps to minimize interference problems.
Purification of solvents by distillation in all-glass systems may be required.
3.2 Interferences by phthalate esters can pose a major problem in pesticide analysis when the EC
detector is used. These compounds generally appear in the chromatogram as large late-eluting
peaks, especially in the 15 and 50% fractions from the Florisil column cleanup. Common flexible
plastics contain varying amounts of phthalates. These phthalates are easily extracted or leached
from such materials during laboratory operations. Cross-contamination of clean glassware occurs
when plastics are handled during extraction steps, especially when solvent-wetted surfaces are
handled. Interferences from phthalates can be minimized by avoiding the use of plastics in the
laboratory. Exhaustive cleanup of reagents and glassware may be required to eliminate
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background phthalate contamination. 3 ’ 4 The interferences from phthalate esters can be avoided by
using a microcoulometric or electrolytic conductivity detector.
3.3 Matrix interferences may be caused by contaminants that are coextracted from the sample. The
extent of matrix interferences will vary considerably from source to source, depending upon the
nature and diversity of the industrial complex or municipality sampled. The cleanup procedure
in Section 11 can be used to overcome many of these interferences, but unique samples may
require additional cleanup approaches to achieve the MDL listed in Table 1.
4. SAFETY
4.1 The toxicity or carcinogenicity of each reagent used in this method has not been precisely defined;
however, each chemical compound must be treated as a potential health hazard. From this
viewpoint, exposure to these chemicals must be reduced to the lowest possible level by whatever
means available. The laboratory is responsible for maint2ining a current awareness file of OSHA
regulations regarding the safe handling of the chemicals specified in this method. A reference file
of material data handling sheets should also be made available to all personnel involved in the
chemical analysis. Additional references to laboratory safety are available and have been
identified for the information of the analyst.
4.2 The following parameters covered by this method have been tentatively classified as known or
suspected human or mammalian carcinogens: aldrin, benzene hexachiorides, chlordane,
heptachlor, PCNB, PCBs, and toxaphene. Primary standards of these toxic materials should be
prepared in a hood.
5. APPARA TUS AND MA TERIALS
5.1 Sampling equipment, for discrete or composite sampling.
5.1.1 Grab-sample bottle: Amber borosiicate or flint glass, 1-L or 1-quart volume, fitted with
screw-caps lined with TFE-fluorocarbon. Aluminum foil may be substituted for TFE if
the sample is not corrosive. If amber bottles are not available, protect samples from
light. The container and cap liner must be washed, rinsed with acetone or methylene
chloride, and dried before use to minimize contamination.
5.1.2 Automatic sampler (optional): Must incorporate glass sample containers for the
collection of a minimum of 250 mL. Sample containers must be kept refrigerated at 4°C
and protected from light during compositing. If the sampler uses a peristaltic pump, a
minimum length of compressible silicone rubber tubing may be used. Before use,
however, the compressible tubing must be thoroughly rinsed with methanol, followed by
repeated rinsings with reagent water to minimize the potential for contamination of the
sample. An integrating flow meter is required to collect flow-proportional composites.
5.2 Glassware. (All specifications are suggested. Catalog numbers are included for illustration only.)
5.2.1 Separatory funnel: 125-mL, 1000-mL, and 2000-mL, with TFE-fluorocarbon stopcock,
ground-glass or TFE stopper.
5.2.2 Drying column: Chromato ranhic column 400 mm long by 19mm ID with coarse-fritted
disc.
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Method 617
5.2.3 Chromatographic column: 400 mm long by 19 mm ID with coarse-fitted disc at bottom
and TFE-fluorocarbon stopcock (Kontes K-420540-0224 or equivalent).
5.2.4 Concentrator tube, Kuderna-Danish: 10-mL, graduated (Kontes K-570050-1025 or
equivalent). Calibration must be checked at the volumes employed in the test. Ground-
glass stopper is used to prevent evaporation of extracts.
5.2.5 Evaporative flask, Kuderna-Danish: 500-mL (Kontes K-570001-0500 or equivalent).
Attach to concentrator tube with springs.
5.2.6 Snyder column, Kuderna-Danish: three-ball macro (Kontes K-503000-0121 or
equivalent).
5.2.7 Vials: Amber glass, 10- to 15-mL capacity with TFE-fluorocarbon-lined screw-cap.
5.3 Boiling chips: Approximately 10/40 mesh. Heat at 400°C for 30 minutes or perform a Soxhiet
extraction with methylene chloride.
5.4 Water bath: Heated, with concentric ring cover, capable of temperature control (±2°C). The
bath should be used in a hood.
5.5 Balance: Analytical, capable of accurately weighing to the nearest 0.0001 g.
5.6 Shaker: Laboratory, reciprocal action.
5.7 Gas chromatograph: Analytical system complete with gas chromatograph suitable for on-column
injection and all required accessories including syringes, analytical columns, gases, detector, and
strip-chart recorder. A data system is recommended for measuring peak areas.
5.6.1 Column 1: 180cm long by 4mm ID glass, packed with 1.5% SP-2250/l.95% SP-240 1
on Supelcoport (100/120 mesh) or equivalent. This column was used to develop the
method performance statements in Section 15. Alternative columns may be used in
accordance with the provisions described in Section 12.1.
5.6.2 Column 2: 180 cm long by 4 mm ID glass, packed with 3% OV-1 on Supelcoport
(100/120 mesh) or equivalent.
5.6.3 Detector: Electron capture. This detector has proven effective in the analysis of
wastewaters for the parameters listed in the scope and was used to develop the method
performance statements in Section 15. Alternative detectors, including a mass
spectrometer, may be used in accordance with the provisions described in Section 121.
6. REAGENTS
6.1 Reagent water: Reagent water is defined as a water in which an interferent is not observed at the
method detection limit of each parameter of interest.
6.2 Acetone, hexane, isooctane, methylene chloride: Pesticide-quality or equivalent.
6.3 Ethyl ether: Nanograde, redistilled in glass if necessary. Must be free of peroxides as indicated
by EM Quant test strips (available from Scientific Products Co., Cat. No. P1126-8, and other
suppliers). Procedures recommended for removal of peroxides are provided with the test strips.
After cleanup, 20 mL ethyl alcohol preservative must be added to each liter of ether.
6.4 Acetonitrile, hexane-saturated: Mix pesticide-quality acetonitrile with an excess of hexane until
equilibrium is established.
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Method 617
6.5 Sodium sulfate: ACS, granular, anhydrous. Heat in a shallow tray at 400°C for a minimum of
4 hours to remove phthalates and other interfering organic substances. Alternatively, heat 16
hours at 450 to 500°C in a shallow tray or Soxhlet extract with methylene chloride for 48 hours.
6.6 Sodium chloride solution, saturated: Prepare saturated solution of NaC1 in reagent water and
extract with hexane to remove impurities.
6.7 Sodium hydroxide solution (iON): Dissolve 40 g ACS grade NaOH in reagent water and dilute
to lOOmL.
6.8 Sulfuric acid solution (1 + I): Slowly add 50 mL H 2 S0 4 (sp. gr. 1.84) to 50 mL of reagent water.
6.9 Mercury: Triple-distill.
6.10 Florisil: PR grade (60/100 mesh). Purchase activated at 675°C and store in dark in glass
container with ground-glass stopper or foil-lined screw-cap. Before use, activate each batch at
least 16 hours at 130°C in a foil-covered glass container.
6.11 Stock standard solutions (1.00 gJpL): Stock standard solutions may be prepared from pure
standard materials or purchased as certified solutions.
6.11.1 Prepare stock standard solutions by accurately weighing approximately 0.0100 gof pure
material. Dissolve the material in pesticide-quality isooctane and dilute to volume in a
10-mL volumetric flask. Larger volumes may be used at the convenience of the analyst.
If compound purity is certified at 96% or greater, the weight may be used without
correctioá to calculate the concentration of the stock standard. Commercially-prepared
stock standards may be used at any concentration if they are certified by the manufacturer
or by an independent source.
6.11.2 Transfer the stock standard solutions into TFE-fiuorocarbon-sealed screw-cap vials.
Store at 4°C and protect from light. Frequently check stock standard solutions for signs
of degradation or evaporation, especially just prior to preparing calibration standards
from them.
6.11.3 Stock standard solutions must be replaced after 6 months, or sooner if comparison with
check standards indicates a problem.
7. CALIBRATION
7.1 Establish gas chromatographic operating parameters equivalent to those indicated in Table 1. The
gas chromatographic system may be calibrated using either the external standard technique
(Section 7.2) or the internal standard technique (Section 7.3).
7.2 External standard calibration procedure.
7.2.1 For each parameter of interest, prepare calibration standards at a minimum of three
concentration levels by adding accurately measured volumes of one or more stock
standards to a volumetric flask and diluting to volume with isooctane. One of the
external standards should be representative of a concentration near, but above, the
method detection limit. The other concentrations should correspond to the range of
concentrations expected in the sample concentrates or should define the working range
of the detector.
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Method 617
7.2.2 Using injections of 1 to 5 L of each calibration standard, tabulate peak height or area
responses against the mass injected. The results can be used to prepare a calibration
curve for each parameter. Alternatively, the ratio of the response to the mass injected,
defined as the calibration factor (CF), may be calculated for each parameter at each
standard concentration. If the relative standard deviation of the calibration factor is less
than 10% over the working range, the average calibration factor can be used in place of
a calibration curve.
7.2.3 The working calibration curve or calibration factor must be verified on each working
shift by the measurement of one or more calibration standards. If the response for any
parameter varies from the predicted response by more than ± 10%, the test must be
repeated using a fresh calibration standard. Alternatively, a new calibration curve or
calibration factor must be prepared for that parameter.
7.3 Internal standard calibration procedure: To use this approach, the analyst must select one or more
internal standards similar in analytical behavior to the compounds of interest. The analyst must
further demonstrate that the measurement of the internal standard is not affected by method or
matrix interferences. Due to these limitations, no internal standard applicable to all samples can
be suggested.
7.3.1 Prepare calibration standards at a minimum of three concentration levels for each
parameter of interest by adding volumes of one or more stock standards to a volumetric
flask. To each calibration standard, add a known constant amount of one or more
internal standards, and dilute to volume with isooctane. One of the standards should be
representative of a concentration near, but above, the method detection limit. The other
concentrations should correspond to the range of concentrations expected in the sample
concentrates, or should define the working range of the detector.
7.3.2 Using injections of 1 to 5 tL of each calibration standard, tabulate the peak height or
area responses against the concentration for each compound and internal standard.
Calculate response factors (RF) for each compound as follows:
Equation 1
RF = ( A )(C )
(A )(C )
where
A = Response for the parameter to be measured
Afr = Response for the internal standard
C, = Concentration of the internal standard, in JLgIL
C, = Concentration of the parameter to be measured, in igIL
If the RF value over the working range is constant, less than 10% relative standard
deviation, the RF can be assumed to be invariant and the average RF may be used for
calculations. Alternatively, the results may be used to plot a calibration curve of
response ratios, A IA against RF.
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Method 617
7.3.3 The working calibration curve or RF must be verified on each working shift by the
measurement of one or more calibration standards. If the response for any parameter
varies from the predicted response by more than ±10%, the test must be repeated using
a fresh calibration standard. Alternatively, a new calibration curve must be prepared for
that compound.
7.4 The cleanup procedure in Section 11 utilizes Florisil chromatography. Florisil from different
batches or sources may vary in adsorptive capacity. To standardize the amount of Florisil which
is used, the use of the lauric acid value is suggested. This procedure’ determines the adsorption
from hexane solution of lauric acid, in milligrams, per gram of Florisil. The amount of Florisil
to be used for each column is calculated by dividing this factor into 110 and multiplying by 20 g.
7.5 Before using any cleanup procedure, the analyst must process a series of calibration standards
through the procedure to validate elution patterns and the absence of interference from the
reagents.
7.6 The multipeak materials included in this method present a special calibration problem.
Recommended procedures for calibration, separation and measurement of PCBs is discussed in
detail in the previous edition of this method. 1 illustrated methods for the calibration and
measurement of chiordane and strobane/toxaphene are available elsewhere. 9
8. QUALITY CONTROL
8.1 Each laboratory using this method is required to operate a formal quality control program. The
minimum requirements of this program consist of an initial demonstration of laboratory capability
and the analysis of spiked samples as a continuing check on performance. The laboratory is
required to maintain performance records to define the quality of data that is generated.
8.1.1 Before performing any analyses, the analyst must demonstrate the ability to generate
acceptable accuracy and precision with this method. This ability is established as
described in Section 8.2.
8.1.2 In recognition of the rapid advances occurring in chromatography, the analyst is
permitted certain options to improve the separations or lower the cost of measurements.
Each time such modifications to the method are made, the analyst is required to repeat
the procedure in Section 8.2.
8.1.3 The laboratory must spike and analyze a minimum of 10% of all samples to monitor
continuing laboratory performance. This procedure is described in Section 8.4.
8.2 To establish the ability to generate acceptable accuracy and precision, the analyst must perform
the following operations.
8.2.1 Select a representative spike concentration for each compound to be measured. Using
stock standards, prepare a quality control check sample concentrate in acetone, 1000
times more concentrated than the selected concentrations.
8.2.2 Using a pipette, add 1.00 mL of the check sample concentrate to each of a minimum of
four 1000-mL aliquots of reagent water. A representative wastewater may be used in
place of the reagent water, but one or more additional aliquots must be analyzed to
determine background levels, and the spike level must exceed twice the background level
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Method 617
for the test to be valid. Analyze the aliquots according to the method beginning in
Section 10.
8.2.3 Calculate the average percent recovery (R), and the standard deviation of the percent
recovery (s), for the results. Wastewater background corrections must be made before
R and s calculations are performed.
8.2.4 Table 2 provides single-operator recovery and precision for many of the organohalide
pesticides. Similar results should be expected from reagent water for all parameters
listed in this method. Compare these results to the values calculated in Section 8.2.3.
If the data are not comparable, review potential problem areas and repeat the test.
8.3 The analyst must calculate method performance criteria and define the performance of the
laboratory for each spike concentration and parameter being measured.
8.3.1 Calculate upper and lower control limits for method performance as follows:
Upper Control Limit (UCL) R + 3s
Lower Control Limit (LCL) R - 3s
where R and s are calculated as in Section 8.2.3. The UCL and LCL can be used to
construct control charts 1 ° that are useful in observing trends in performance.
8.3.2 The laboratory must develop and maintain separate accuracy statements of laboratory
performance for wastewater samples. An accuracy statement for the method is defined
as R ± s. The accuracy statement should be developed by the analysis of four aliquots
of wastewater as described in Section 8.2.2, followed by the calculation of R and s.
Alternatively, the analyst may use four wastewater data points gathered through the
requirement for continuing quality control in Section 8.4. The accuracy statements
should be updated regularly.’ 0
8.4 The laboratory is required to collect in duplicate a portion of their samples to monitor spike
recoveries. The frequency of spiked sample analysis must be at least 10% of all samples or one
spiked sample per month, whichever is greater. One aliquot of the sample must be spiked and
analyzed as described in Section 8.2. If the recovery for a particular parameter does not fall
within the control limits for method performance, the results reported for that parameter in all
samples processed as part of the same set must be qualified as described in Section 13.3. The
laboratory should monitor the frequency of data so qualified to ensure that it remains at or
below 5%.
8.5 Before processing any samples, the analyst must demonstrate through the analysis of a l-L aliquot
of reagent water that all glassware and reagent interferences are under control. Each time a set
of samples is extracted or there is a change in reagents, a laboratory reagent blank must be
processed as a safeguard against laboratory contamination.
8.6 It is recommended that the laboratory adopt additional quality assurance practices for use with this
method. The specific practices that are most productive depend upon the needs of the laboratory
and the nature of the samples. Field duplicates may be analyzed to monitor the precision of the
sampling technique. When doubt exists over the identification of a peak on the chromatogram,
confirmatory techniques such as gas chromatography with a dissimilar column, specific element
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Method 617
detector, or mass spectrometer must be used. Whenever possible, the laboratory should perform
analysis of quality control materials and participate in relevant performance evaluation studies.
9. SAMPLE CoLLECTiON, PRESERVA liON, AND HANDLING
9.1 Grab samples must be collected in glass containers. Conventional sampling practice&’ should be
followed; however, the bottle must not be prerinsed with sample before collection. Composite
samples should be collected in refrigerated glass containers in accordance with the requirements
of the program. Automatic sampling equipment must be as free as possible of plastic and other
potential sources of contamination.
9.2 The samples must be iced or refrigerated at 4°C from the time of collection until extraction.
Chemical preservatives should not be used in the field unless more than 24 hours will elapse
before delivery to the laboratory. If the samples will not be extracted within 48 hours of
collection, the sample should be adjusted to a pH range of 6.0 to 8.0 with sodium hydroxide or
sulfuric acid. Record the volume of acid or base used.
9.3 All samples must be extracted within 7 days and completely analyzed within 40 days of extraction.
10. SAMPLE EXTRACTiON
10.1 Mark the water ineniscus on the side of the sample bottle for later determination of sample
volume. Pour the entire sample into a 2-L separatory funnel.
10.2 Add 60 mL 15% methylene chloride in hexane (V:V) to the sample bottle, seal, and shake 30
seconds to rinse the inner walls. Transfer the solvent to the separatory funnel and extract the
sample by sh2king the funnel for 2 minutes with periodic venting to release excess pressure.
Allow the organic layer to separate from the water phase for a minimum of 10 minutes. If the
emulsion interface between layers is more than one-third the volume of the solvent layer, the
analyst must employ mechanical techniques to complete the phase separation. The optimum
technique depends upon the sample, but may include stirring, filtration of the emulsion through
glass wool, cantrifugation, or other physical methods. Drain the aqueous phase into a 1000-mL
Erlenmeyer flask and collect the extract in a 250-mL Erlenmeyer flask. Return the aqueous phase
to the separatory funnel.
10.3 Addasecond6O -mLvolumeofl5%methylenechlorideinhexanetothesamplebottleafldrePeat
the extraction procedure a second time, combining the extracts in the 250-mL Erlenmeyer flask.
Perfbrm a third extraction in the same m2nner.
10.4 Assemble a Kuderna-Danish (K-D) concentrator by atththing a 10-mL concentrator tube to a 500-
mL evaporative flask. Other concentration devices or techniques may be used in place of the K-D
if the requirements of Section 8.2 are met.
10.5 Pour the combined extract through a drying column containing about 10 cm of anhydrous sodium
sulfate, and collect the extract in the K-D concentrator. Rinse the Erlenmeyer flask and column
with 20 to 30 mL of hexane to complete the quantitative transfer.
10.6 Add one or two clean boiling chips to the evaporative flask and att2ch a three-ball Snyder column.
Prewet the Snyder column by adding about I mL methylene chloride to the top. Place the K-D
apparatus on a hot water bath, 80 to 85°C, so that the concentrator tube is partially immersed in
148
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Method 617
the hot water, and the entire lower rounded surface of the flask is bathed with hot vapor. Adjust
the vertical position of the apparatus and the water temperature as required to complete the
concentration in 15 to 20 minutes. At the proper rate of distillation, the balls of the column will
actively chatter but the chambers will not flood with condensed solvent. When the apparent
volume of liquid reaches 1 mL, remove the K-D apparatus and allow it to drain and cool for at
least 10 minutes.
10.7 Remove the Snyder column and rinse the flask and its lower joint into the concentrator tube with
1 to 2 mL of hexane and adjust the volume to 10 mL. A 5-mL syringe is recommended for this
operation. Stopper the concentrator tube and store refrigerated if further processing will not be
performed immediately. If the extracts will be stored longer than two days, they should be
transferred to PTFE-sealed screw-cap bottles. If the sample extract requires no further cleanup,
proceed with gas chromatographic analysis. If the sample requires cleanup, proceed to
Section 11.
10.8 Determine the original sample volume by refilling the sample bottle to the mark and transferring
the water to a 1000-mL graduated cylinder. Record the sample volume to the nearest 5 mL.
11. CLEANUP AND SEPARATION
1 1.1 Cleanup procedures may not be necessary for a relatively clean sample matrix. The cleanup
procedure recommended in this method has been used for the analysis of various industrial and
municipal effluents. If particular circumstances demand the use of an alternative cleanup
procedure, the analyst must determine the elution profile and demonstrate that the recovery of
each compound of interest for the cleanup procedure is no less than 85%.
1 1.2 Acetonitrile partition: The following acetonitrile partitioning procedure may be used to isolate fats
and oils from the sample extracts. This procedure is applicable to all of the parameters in this
method except mirex.
11.2.1 Quantitatively transfer the previously concentrated extract to a 125-mL separatory funnel
with enough hexane to bring the final volume to 15 mL. Extract the sample four times
by shaking vigorously for 1 minute with 30-mL portions of hexane-saturated acetonitrile.
11.2.2 Combine and transfer the acetonitrile phases to a 1-L separatory funnel and add 650 mL
of reagent water and 40 mL of saturated sodium chloride solution. Mix thoroughly for
30 to 45 seconds. Extract with two l00-mL portions of hexane by vigorously shaking
for 15 seconds.
11.2.3 Combine the hexane extracts in a 1-L separatory funnel and wash with two 100-mL
portions of reagent water. Discard the water layer and pour the hexane layer through a
drying column containing 7 to 10 cm of anhydrous sodium sulfate into a 500-mL K-D
flask equipped with a l0-mL concentrator tube. Rinse the separatory funnel and column
with three 10-mL portions of hexane.
11.2.4 Concentrate the extracts to 6 to 10 mL in the K-D as directed in Section 10.6. Adjust
the extract volume to 10 mL with hexane.
11.2.5 Analyze by gas chromatography unless a need for further cleanup is indicated.
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Method 617
11.3 Florisil column cleanup: The following Florisil column cleanup procedure has been demonstrated
to be applicable to most of the organochlorine pesticides and PCBs listed in Table 3. It should
also be applicable to the cleanup of extracts for PCNB, strobane, and triuluralin.
11.3.1 Add a weight of Florisil (nominally 20 g), predetermined by calibration (Section 7.4
and 7.5), to a chromatographic column. Settle the Florisil by tapping the column. Add
anhydrous sodium sulfate to the top of the Florisil to form a layer I to 2 cm deep. Add
60 mL of hexane to wet and rinse the sodium sulfate and Florisil. Just prior to exposure
of the sodium sulfate to air, stop the elution of the hexane by closing the stopcock on the
chromatography column. Discard the eluate.
11.3.2 Adjust the sample extract volume to 10 mL with hexane and transfer it from the K-D
concentrator tube to the Florisil column. Rinse the tube twice with 1 to 2 mL hexane,
adding each rinse to the column.
11.3.3 Place a 500-mL K-D flask and clean concentrator tube under the chromatography
column. Drain the column into the flask until the sodium sulfate layer is nearly exposed.
Elute the column with 200 mL of 6% ethyl ether in hexane (V/V) (Fraction 1) using a
drip rate of about 5 mL/min. Remove the K-D flask and set aside for later
concentration. Elute the column again, using 200 mL of 15% ethyl ether in hexane
(WV) (Fraction 2), into a second K-D flask. Perform a third elution using 200 mL of
50% ethyl ether in hexane (WV) (Fraction 3) into a separate K-D flask. The elution
patterns for the pesticides and PCBs are shown in Table 3.
11.3.4 Concentrate the eluates by standard K-D techniques (Section 10.6), using the water bath
at about 85°C. Adjust final volume to 10 mL with hexane. Analyze by gas
chromatography.
11.4 Removal of sulfur: Elemental sulfur will elute in Fraction I of the Florisil cleanup procedure.
If a large amount of sulfur is present in the extract, it may elute in all fractions. If so, each
fraction must be further treated to remove the sulfur. This procedure cannot be used with
heptachior; eKIoSUlfanS, or endrin aldehyde.
11.4.1 Pipette 1.00 mL of the concentrated extract into a clean concentrator tube or a vial with
a TFE-fluorocarbon seal. Add I to 3 drops of mercury and seal.
11.4.2 Agitate the contents of the vial for 15 to 30 seconds.
11.4.3 Place the vial in an upright position on a reciprocal laboratory shaker and shake for up
to 2 hours.
11.4.4 If the mercury appears shiny alter this treatment, analyze the extract by gas
chromatography. If the mercury is black, decant the extract into a clean vial and repeat
the cleanup with fresh mercury.
12. GAs CHROMATOGRAPHY
12.1 Table I summarizes the recommended operating conditions for the gas chromatograph. Included
in this table are estimated retention times and method detection limits that can be achieved by this
method. Other packed columns, chromatographic conditions, or detectors may be used if the
requirements of Section 8.2 are met. Capillary (open-tubular) columns may also be used if the
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Method 617
relative standard deviations of responses for replicate injections are demonstrated to be less
than 6% and the requirements of Section 8.2 are met.
12.2 Calibrate the system daily as described in Section 7.
12.3 If the internal standard approach is being used, add the internal standard to sample extracts
immediately before injection into the instrument. Mix thoroughly.
12.4 Inject 1 to 5 zL of the sample extract using the solvent-flush technique.’ 2 Record the volume
injected to the nearest 0.05 iL, and the resulting peak size in area or peak height units. An
automated system that consistently injects a constant volume of extract may also be used.
Multipeak materials present a special analytical problem beyond the scope of this discussion.
Illustrated procedures for calibration and measurement are available for PCBs’ and pesticides. 9
12.5 The width of the retention-time window used to make identifications should be based upon
measurements of actual retention-time variations of standards over the course of a day. Three
times the standard deviation of a retention time can be used to calculate a suggested window size
for a compound. However, the experience of the analyst should weigh heavily in the
interpretation of chromatograms.
12.6 If the response for the peak exceeds the working range of the system, dilute the extract and
reanalyze.
12.7 If the measurement of the peak response is prevented by the presence of interferences, further
cleanup is required.
13. CALCULATIONS
13.1 Determine the concentration of individual compounds in the sample.
13.1.1 If the external standard calibration procedure is used, calculate the amount of material
injected from the peak response using the calibration curve or calibration factor in
Section 7.2.2. The concentration in the sample can be calculated as follows:
Equation 2
( A)(V )
Concentration, gIL = (V.)(V)
where
A = Amount of material injected, in ng
V 3 = Voiwne of extract injected, in 1 zL
= Voiwne of total extract, in 1 uL
V , = Volume of water extracted, in niL
13.1.2 If the internal standard calibration procedure was used, calculate the concentration in the
sample using the response factor (RF) determined in Section 7.3.2 as follows:
151
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Method 617
Equation 3
Concentration, gIL = (A)(!, )
(A 1 )(RF)(V 0 )
where:
A, = Response for parameter to be measured.
4, = Response for the internal standard.
I , = Amount of internal standard added to each extract, in gig.
= Volume of water extracted, in liters.
13.2 Report results in micrograms per liter without correction for recovery data. When duplicate and
spiked samples are analyzed, report all data obtained with the sample results.
13.3 For samples processed as part of a set where the laboratory spiked sample recovery falls outside
of the control limits in Section 8.3, data for the affected parameters must be labeled as suspect.
14. GC/MS COavRRM4 T1ON
14.1 It is recommended that GCIMS techniques be judiciously employed to support qualitative
compound idenifications made with this method. The mass spectrometer should be capable of
scanning the mass range from 35 amu to a mass 50 amu above the molecular weight of the
compound. The instrument must be capable of scanning the mass range at a rate to produce at
least five scans per peak but not to exceed seven per scan utilizing a 70 V (nominal) electron
energy in the electron impact ionization mode. A GC to MS interface constructed of all glass or
glass-lined materials is recommended. A computer system should be interfaced to the mass
spectrometer that allows the continuous acquisition and storage on machine-readable media of all
mass spectra obtained throughout the duration of the chromatographic program.
14.2 Gas chromatographiC columns and conditions should be selected for optimum separation and
performance. The conditions selected must be compatible with standard GCJMS operating
practices. Chromatographic tailing factors of less than 5.0 must be achieved.’ 3
14.3 At the beginning of each day that confirmatory analyses are to be performed, the GCIMS system
must be checked to see that all decafluorotriphenyl phosphine (DFTPP) performance criteria are
achieved.’ 4
14.4 To confirm an identification of a compound, the background-corrected mass spectrum of the
compound must be obtained from the sample extract and compared with a mass spectrum from
a stock or calibration standard analyzed under the same chromatographic conditions. It is
recommended that at least 25 ng of material be injected into the GCIMS. The criteria below must
be met for qualitative confirmation.
14.4.1 All ions that are present above 10% relative abundance in the mass spectrum of the
standard must be present in the mass spectrum of the sample with agreement to ±10%.
For example, if the relative abundance of an ion is 30% in the mass spectrum of the
standard, the allowable limits for the relative abundance of that ion in the mass spectrum
for the sample would be 20% to 40%.
152
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Method 617
14.4.2 The retention time of the compound in the sample must be within 6 seconds of the same
compound in the standard solution.
14.4.3 Compounds that have very similar mass spectra can be explicitly identified by GC/MS
only on the basis of retention time data.
14.5 Where available, chemical ionization mass spectra may be employed to aid in the qualitative
identification process.
14.6 Should these MS procedures fail to provide satisfactory results, additional steps may be taken
before reanalysis. These may include the use of alternate packed or capillary GC columns or
additional cleanup (Section 11).
15. ME HOD PERFORMANCE
15.1 The method detection limit (MDL) is defined as the minimum concentration of a substance that
can be measured and reported with 99% confidence that the value is above zero. 15 The MDL
concentrations listed in Table 1 were obtained using reagent water. 16
15.2 In a single laboratory, Susquehanna University, using spiked tap water samples, the average
recoveries presented in Table 2 were obtained. The standard deviation of the percent recovery
is also included in Table 2.16
153
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Method 617
References
1. “Methods for Benzidine, Chlorinated Organic Compounds, Pentachiorophenol and Pesticides in
Water and Wastewater,” U.S. Environmental Protection Agency, Environmental Monitoring and
Support Laboratory, Cincinnati, Ohio, September 1978.
2. ASTM Annual Book of Standards, Part 31, D3694, “Standard Practice for Preparation of Sample
Containers and for Preservation, “American Society for Testing and Materials, Philadelphia, PA,
p. 679, 1980.
3. Giam, D.S., Chan, H.S. and Nef, G.S., “Sensitive Method for Determination of Phthalate Ester
Plasticizers in Open-Ocean Biota Samples,” Analytical Ozemistiy, 47, 2225, (1975).
4. Giazn, C.S., and Chan, H.S., “Control of Blanks in the Analysis of Phthalates in Air and Ocean
Biota Samples, National Bureau of Standards (U.S.), Special Publication 442, pp. 701-708, 1976.
5. “Carcinogens - Working with Carcinogens,” Department of Health, Education, and Welfare,
Public Health Service, Center for Disease Control, National Institute for Occupational Safety and
Health, Publication No. 77-206, Aug. 1977.
6. “OSHA Safety and Health Standards, General Industry,” (29 CFR 1910), Occupational Safety and
Health Administration, OSHA 2206, (Revised, January 1976).
7. “Safety in Academic Chemistry Laboratories,” American Chemical Society Publication,
Committee on Chemical Safety, 3rd Edition, 1979.
8. ASTM Annual Book of Standards, Part 31, D3086, Appendix X3, “Standardization of Florisil
Column by Weight Adjustment Based on Adsorption of Lauric Acid,” American Society for
Testing and Materials, Philadelphia, PA, p. 765, 1980.
9. “Pesticide Analytical Manual Volume 1,” U.S. Department of Health and Human Services, Food
and Drug Administration.
10. “Handbook for Analytical Quality Control in Water and Wastewater Laboratories,” EPA-600/4-
79-019, U. S. Environmental Protection Agency, Environmental Monitoring and Support
Laboratory - Cincinnati, Ohio, March 1979.
11. ASTM Annual Book of Standards, Part 31, D3370, “Standard Practice for Sampling Water,”
American Society for Testing and Materials, Philadelphia, PA, p. 76, 1980.
12. Burke, J. A., “Gas Chromatography for Pesticide Residue Analysis; Some Practical Aspects,”
Journal of the Association of Official Analytical Chemists, 48, 1037 (1965).
13. McNair, H.M. and Bonelli, E. J., “Basic Chromatography,” Consolidated Printing, Berkeley,
California, p. 52, 1969.
154
-------
Method 617
References (cont.)
14. Eichelberger, J.W., Harris, L.E., and Budde, W.L. “Reference Compound to Calibrate Ion
Abundance Measurement in Gas Chromatography-Mass Spectrometry,” Analytical Chemistry, 47,
995 (1975).
15. Glaser, l.A. et.al, “Trace Analysis for Wastewaters,” Environmental Science & Technology, 15,
1426 (1981).
16. McGrath, T. F., “Recovery Studies of Pesticides From Surface and Drinking Waters,” Final
Report for U.S. EPA Grant R804294, Environmental Monitoring and Support Laboratory,
Cincinnati, Ohio 45268.
155
-------
Method 617
Table 1. Gas Chromotagraphy of Pesticides and PCBs
Method
Detection
Retention Time (mini Limit
Parameter Column 1 Column 2 (pg/Li
Aidrin 2.40 4.10 0.009
a-BHC 1.35 1.82 0.004
-BHC 1.90 1.97 ND
6BHC 2.15 2.20 ND
y-BHC 1.70 2.13 0.002
Captan 6.22 5.00 ND
Carbophenothion 10.9 10.90 ND
4,4’-DDD 7.83 9.08 0.012
4,4’-DDE 5.13 7.15 0.004
4,4’-DDT 9.40 11.75 0.032
Dichloran 1.85 2.01 ND
Dicofol 2.86 4.59 ND
Diekbin 545 7.23 0.011
Endosulfan l 4.50 6.20 0.11
Endosulfan U 8.00 8.28 0.17
Endosulfan sulfate 14.22 10.70 ND
Endrin 6.55 8.19 ND
Endrinaldebyde 11.82 9.30 ND
Hq,tachlor 2.00 3.35 0.004
Heptachior epoxide 3.50 5.00 0.003
Isodrin 3.00 4.83 ND
Methoxychlor 18.20 26.60 0.176
Mirex 14.60 15.50 0.015
PCNB 1.63 2.01 0.002
Tritluralin 0.94 1.35 0.013
*For multipeak materials, see Figures 2 to 10 for chromatographic conditions and retention patterns.
ND = Not Determined
Column 1 conditions: Supelcoport(1001120 mesh) coated with 1.5% SP-225011.95% SP-2401 in a 1.8 m
long by 4 mm ID glass column with 95% argon/5% methane carrier gas at a flow rate of 60 mL/min.
Column temperature: isothermal at 200°C. An electron capture detector was used with this column to
determine the MDL.
Column 2 conditions: Supelcoport (100/120 mesh) coated with 3% OV-1 packed in a 1.8 m long by
4 mm ID glass column with 95% argon/5% methane carrier gas at a flow rate of 60 mL/min. Column
temperature: isothermal at 200°C.
156
-------
Method 617
Table 2. Single-Operator Accuracy and Precision for Tap Water
Average Standard Spike Number
Percent Deviation Range of
Parameter Recovery (%) (pg/L) Analyses
Aidrin 78.1 5.4 0.03-3.0 21
ô-BHC 95.3 8.9 0.01-1.0 21
y-BHC 95.1 7.2 0.01-1.0 21
4,4’-DDD 94.4 5.0 0.08-8.0 21
4,4’-DDE 89.8 3.7 0.05-5.0 21
4,4’-DDT 91.0 4.5 0.2-20 21
Dieldrin 98.2 4.9 0.06-6.0 21
Endosulfan I 101.0 7.6 0.05-5.0 21
Endosulfan II 92.9 4.8 0.09-9.0 21
Heptachlor 84.4 6.4 0.02-2.0 21
Heptachior epoxide 93.7 3.9 0.03-3.0 21
Methoxychlor 96.6 6.7 0.6-60 21
Mirex 89.1 4.8 0.2-20 21
PCNB 82.6 6.2 0.01-1.0 21
Trifluralin 94.3 10.5 0.03-3.0 21
157
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Method 617
Table 3. Distribution and Recovery of Chlorinated Pesticides and PCBs Using
Florisil Column Chromatography
Percent Recovery by Fraction
Parameter No. I No. 2 No. 3
A ldrin 100
a-BHC 100
$-BHC 97
b-BHC 98
y-BHC 100
Captan + +
Carbofenthion 100
Chiordane 100
4,4’-DDD 99
4,4’-DDE 98
4,4’-DDT 100
Dicofol + +
Dieldrin 0 100
Endosulfan 11 37 64 91
Endosulfan U 0 7 106
Endosulfan sulfate 0 0
Endrin 4 96
Endrin aldehyde 0 68 26
Heptachlor 100
Heptachior epoxide 100
Isodrin 100
Methoxychlor 100
Mirex 100
Perthane 100
Toxaphene 96
PcB-1016 97
PcB-1221 97
PCB-1232 95
PCB-1242 97
PCB-1248 103
PcB-1254 90
PcB-126o 95
+Compound occurs in both 6% and 15% fractions.
Florisil eluate composition by fraction:
Fraction I - 200 mL of 65 ethyl ether in hexane
Fraction 2- 200 mL of 15% ethyl ether in hexane
Fraction 3-200 mL of 50% ethyl ether in hexane
158
-------
Method 617
Column: 1.5% SP-2250+
1.95% SP-2401 on Supelcoport
Temperature: 200°C
Detector: Electron Capture
C.)
z
‘0 &
I W
F-
w ô
c
= (U
0 )
w
I 1 I I I I
0 4.0 8.0 12.0 16.0
Retention lime (minutes)
Figure 1. Gas Chromatogram of Pesticides.
159
-------
A *I 617
Cokimn: 1.5% SP-2250+
1.95% SP-2401 on Supelcoport
Terr erature: 200°C.
Detector. Electron Capture
I
AJL
I I I I I I I I
0 4.0 8.0 12.0 16.0
Retention Thie (minutes)
Figure 2. Gas Chromatogram of Chiordane
160
-------
Method 617
Retention Time (minutes)
Figure 3. Gas Chromatogram of Toxaphene.
Column: 1.5% SP-2250+
1.95% SP2401 on Supelcoport
Temperature: 200°C
Detector: Electron Capture
0 2.0
1 I I I I I 1 I 1 I I I
6.0 10.0 14.0 18.0 22.0 26.0
*52.002.41
161
-------
Method 617
Column: 1.5% SP-2250+ 1.95% SP-2401 on Supelcopoil
TerTperature: 160°C
Detector: Electron Capture
2.0
6.0
10.0
14.0 18.0 22.0
Retention TIme (minutes)
Figure 4. Gas Chromatogram of PCB-1 016.
-------
Method 617
Column: 1.5% SP-2250+ 1.95% SP-2401 on Supelooport
Temperature: 160°C
Detector: Electron Capture
0 2.0 6.0 10.0 14.0 18.0 22.0
Retention Time (minutes)
A 43
Figure 5. Gas Chromatogram of PCB-1 221.
163
-------
M.thod 617
Figure 6. Gas Chromatogram of PCB-1 232.
Column: 1.5% SP-2250+ 1.95% SP-2401 on Supelcopoit
Tervçerature: 160°C
Detector: Electron Capture
0 2.0 6.0 10.0 14.0 18.0 22.0
Thn. )
22.0
164
-------
Method 617
Retention Time (minutes)
Figure 7. Gas Chromatogram of PCB-1 242
Column: 1.5% SP-2250 +1.95% SP-2401
on Supelcoport
Terr erature: 160°C.
Detector: Electron Capture
A 45
165
-------
Molhod 617
I I I I I I I I I I F
o 2.0 8.0 10.0 14.0 18.0 22.0 26.0
R .nUan Time (mlni*e.)
Cobnm: 1.5% SP-2250 +1.95% SP-2401 on
Temperature: 160°C.
Detector: Electron Capture
Figure 8. Gas Chromatogram of PCB-1 248
-------
Method 617
I I
I I I I
U I
I I I
2.0
6.0
10.0
14.0
18.0
22.0
Retention Time (minutes)
Figure 9. Gas Chromatogram of PCB-1 254.
Column: 1.5% SP-2250+ 1.95% SP-2401 on Supelcoport
Ten erature: 200°C
Detector: Electron Capture
A52 .002-47
167
-------
Method 617
Cokimn: 1.5% SP-2250+ 1.95% SP-2401 on Supelcoport
Ten erature: 200°C
Detector: Electron Capture
1 I I I I I
6.0 10.0 14.0 18.0 22.0
I I
26.0
Retention Time (mkwt.s) A CO2 .48
0 2.0
Figure 10. Gas Chromatogram of PCB-1260.
-------
Method 618
The Determination of Volatile
Pesticides in Municipal and
Industrial Waste water
-------
Method 618
The Determination of Volatile Pesticides in Municipal and Industrial
Waste water
1. SCOPE AND APPLICATION
1.1 This method covers the determination of certain volatile pesticides. The following parameters can
be determined by this method:
Parameter CAS No.
Chioropicrin 76-06-2
Ethylene dibromide 106-934
1.2 This is a gas chromatographic (GC) method applicable to the determination of the compounds
listed above in municipal and industrial discharges.
1.3 The method detection limit (MDL, defmed in Section 15) for each compound is listed in Table 1.
The MDL for a specific wastewater may differ from those listed, depending upon the nature of
interferences in the sample matrix.
1.4 This method is restricted to use by or under the supervision of analysts experienced in the use of
gas chromatography and in the interpretation of gas chromatograms. Each analyst must
demonstrate the ability to generate acceptable results with this method using the procedure
described in Section 8.2.
1.5 When this method is used to analyze unfamiliar samples for any or all of the compounds above,
compound identifications should be supported by at least one additional qualitative technique.
This method describes analytical conditions for a second gas chromatographic column that can be
used to confirm measurements made with the primary column. Section 14 provides gas
chromatograph/mass spectrometer (GCIMS) criteria appropriate for the qualitative confirmation
of compound identifications.
2. SUMMARY OF METHOD
2.1 A measured volume of sample, 20 mL, is extracted with cyclohexane. The cyclohexane extract
is analyzed with no additional treatment. Gas chromatographic conditions are described which
permit the separation of the compounds in the extract and their measurement by an electron
capture detector.
3. INTERFERENCES
3.1 Method interferences may be caused by contaminants in solvents, reagents, glassware, and other
sample-processing apparatus that lead to discrete artifacts or elevated baselines in
gaschromatograms. All reagents and apparatus must be routinely demonstrated to be free from
interferences under the conditions of the analysis by running laboratory reagent blanks as
described in Section 8.5.
171
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Method 618
3.1.1 Glassware must be scrupulously cleaned. 1 Clean all glassware as soon as possible after
use by thoroughly rinsing with the last solvent used in it. Follow by washing with hot
water and detergent and thorough rinsing with tap and reagent water. Drain dry, and
heat in an oven or muffle furnace at 400°C for 15 to 30 minutes. Do not heat volumetric
ware. Thorough rinsing with acetone may be substituted for the heating. After drying
and cooling, seal and store glassware in a clean environment to prevent any accumulation
of dust or other contaminants. Store inverted or capped with aluminum foil.
3.1.2 The use of high-purity reagents and solvents helps to minimi7e interference problems.
Purification of solvents by distillation in all-glass systems may be required.
3.2 Matrix interferences may be caused by contaminants that are coextracted from the sample. The
extent of matrix interferences will vary considerably from source to source, depending upon the
nature and diversity of the industrial complex or municipality being sampled. Some samples may
require a cleanup approach to achieve the MDL listed in Table 1.
4. SAFETY
4.1 The toxicity or carcinogenicity of each reagent used in this method has not been precisely defined;
however, each chemical compound should be treated as a potential health hazard. Chloropicrin
produces severe sensory irritation in upper respiratory passages. It has strong lacrimatory
properties and produèes increased sensitivity after frequent exposures. Taken orally, chloropicrin
causes severe nausea, vomiting, colic, and diarrhea. Chioropicrin is a potent skin irritant.
Ethylene dibromide liquid on the skin causes blisters if evaporation is delayed. Inhalation of
ethylene dibromide causes delayed pulmonary lesions. Prolonged exposure may also result in
liver and kidney injury. Exposure to these chemicals must be reduced to the lowest possible level
by whatever means available. The laboratory is responsible for maintaining a current awareness
file of OSHA regulations regarding the safe handling of the chemicals specified in this method.
A reference file of material data handling sheets should also be made available to all personnel
involved in the chemical analysis. Additional references to laboratory safety are available and
have been identified for the information of the analyst.
5. APPARA TUS AND MA TEPJALS
5.1 Sampling equipment, for discrete sampling.
5.1.1 Vial: 25-anL capacity or larger, equipped with a screw-cap with hole in center (Pierce
No. 13075 or equivalent). Detergent wash, rinse with tap and distilled water, and dry
at 105°C before use.
5.1.2 Sq,tum: PTFE-faced silicone (Pierce No. 12722 or equivalent). Detergent wash, rinse
with tap and distilled water, and dry at 105°C before use.
5.2 Glassware (all specifications are suggested).
5.2.1 Centrifuge tube: 40-inL, with screw-cap lined with FI’FE.
5.2.2 Pipette: 4-mL graduated.
5.2.3 Graduated cylinder: 25-mL.
5.2.4 Volumetric flask: 1O-mL, ground-glass stoppered.
172
-------
Method 618
5.3 Balance: Analytical, capable of accurately weighing to the nearest 0.0001 g.
5.4 Gas chromatograph: Analytical system complete with gas chromatograph suitable for on-column
injection and all required accessories including syringes, analytical columns, gases, detector, and
strip-chart recorder. A data system is recommended for measuring peak areas.
5.4.1 Column 1: 180 cm long by 2 mm ID glass, packed with 1% SP-1000 on Carbopak B
(60/80 mesh) or equivalent. This column was used to develop the method performance
statements in Section 15. Alternative columns may be used in accordance with the
provisions described in Section 11.1.
5.4.2 Column 2: 180 cm long by 2 mm ID glass, packed with 30% OV-17 on Gas Chrom Q
(100/120 mesh) or equivalent.
5.4.3 Detector: electron capture. This detector has proven effective in the analysis of
wastewaters for the compounds listed in the scope and was used to develop the method
performance statements in Section 15. Alternative detectors, including a mass
spectrometer, may be used in accordance with the provisions described in Section 12.1.
6. REAGENTS
6.1 Reagent water: Reagent water is defined as a water in which an interferent is not observed at the
method detection limit of each compound of interest.
6.2 Cyclohexane: Pesticide-quality or equivalent. Because of the frequent occurrence of contaminants
in solvents, interfering with electron capture several lots of solvent, or a different solvent, e.g.,
hexane, heptane, or isooctane, may have to be analyzed to find a suitable extraction solvent.
6.3 Sodium hydroxide: 6N in distilled water.
6.4 Sulfuric acid: 6N in distilled water.
6.5 Stock standard solutions (20 mg/mi): Stock standard solutions can be prepared from pure standard
materials or purchased as certified solutions. Prepare stock solutions in cyclohexane using assayed
liquids.
6.5.1
Place about 9.5 mL of pesticide-quality cyclohexane in a 10-mL volumetric flask. Allow
the flask to stand, unstoppered, for about 5 minutes or until all cyclohexane-wetted
surfaces have dried. Weigh the flask to the nearest 0.1 mg. Using a 250- &L syringe,
immediately add 121 iL of chloropicrin (d 4 = 1.66) and/or 92 iL of ethylene
dibromide (d 4 z = 2.18). The liquid must fall directly into the cyclohexane without
contacting the neck of the flask. Reweigh, dilute to volume, stopper, and mix by
inverting the flask several times. Calculate the concentration in milligrams per
milliliter (mg/mL) from the net gain in weight. Larger volumes can be used at the
convenience of the analyst. If compound purity is certified at 96% or greater, the weight
can be used without correction to calculate the concentration of the stock standard.
Commercially prepared stock standards can be used at any concentration if they are
certified by the manufacturer or by an independent source.
6.5.2 Transfer the stock standard solutions into PTFE-sealed screw-cap bottles. Store at 4°C
and protect from light. Frequently check stock standard solutions for signs of
degradation or evaporation, especially just prior to preparing calibration standards from
them.
173
-------
Method 618
6.5.3 Stock standard solutions must be replaced after 6 months, or sooner if comparison with
check standards indicates a problem.
7. CALIBRATION
7.1 Establish gas chromatographic operating parameters equivalent to those indicated in Table 1. The
gas chromatographic system may be calibrated using either the external standard technique
(Section 7.2) or the internal standard technique (Section 7.3).
7.2 External standard calibration procedure.
7.2.1 For each compound of interest, prepare calibration standards at a minimum of three
concentration levels by adding accurately measured volumes of one or more stock
standards to a volumetric flask and diluting to volume with cyclohexane. One of the
external standards should be representative of a concentration near, but above, the
method detection limit. The other concentrations should correspond to the range of
concentrations expected in the sample concentrates or should define the working range
of the detector.
7.2.2 Using injections of 1 to 5 iL of each calibration standard, tabulate peak height or area
responses against the mass injected. The results can be used to prepare a calibration
curve for eah parameter. Alternatively, the ratio of the response to the mass injected,
defined as the calibration factor (CF), can be calculated for each compound at each
standard concentration. If the relative standard deviation of the calibration factor is less
than 10% over the working range, the average calibration factor can be used in place of
a calibration curve.
7.2.3 The working calibration curve or calibration factor must be verified on each working
shift by the measurement of one or more calibration standards. If the response for any
compound varies from the predicted response by more than ± 10%, the test must be
repeated using a fresh calibration standard. Alternatively, a new calibration curve or
calibration factor must be prepared for that parameter.
73 Internal standard calibration procedure: To use this approach, the analyst must select one or more
internal standards similar in analytical behavior to the compounds of interest. The analyst must
farther demonstrate that the measurement of the internal standard is not affected by method or
matrix int ferences. Due to these limitations, no internal standard applicable to all samples can
be specified; however, bronioform ha been shown to be satisfactory in some cases.
7.3.1 Prepare calibration standards at a minimum of three concentration levels for each
compound of interest by adding volumes of one or more stock standards to a volumetric
flask . To each calibration standard, add a known constant amount of one or more
Internal standards, and dilute to volume with cycloliexane. One of the standards should
be representative of a concentration near, but above, the method detection limit. The
other concentrations should correspond to the range of concentrations expected in the
sample concentrates or should define the working range of the detector.
7.3.2 Using injections of 1 to 5 1 iL of each calibration standard, tabulate the peak height or
area responses against the concentration for each compound and internal standard.
Calculate response factors (RF) for each compound as follows:
174
-------
Method 618
Equation 1
( A,)(C , )
(As) (C,)
where
A, = Response for the parameter to be measured
A, = Response for the internal standard
C = Concentration of the internal standard, in p.gIL
C, = Concentration of the parameter to be measured, in g/L
If the RF value over the working range is constant, less than 10% relative standard
deviation, the RF can be assumed to be invariant and the average RF can be used for
calculations. Alternatively, the results can be used to plot a calibration curve of response
ratios, AJA against RF.
7.3.3 The working calibration curve or RF must be verified on each working shift by the
measurement of one or more calibration standards. If the response for any compound
varies from the predicted response by more than ±10%, the test must be repeated using
a fresh calibration standard. Alternatively, a new calibration curve must be prepared for
that compound.
7.4 Before using any cleanup procedure, the analyst must process a series of calibration standards
through the procedure to validate elution patterns and the absence of interferences from the
reagents.
8. (2uAuTv CONTROL
8.1 Each laboratory using this method is required to operate a formal quality control program. The
minimum requirements of this program consist of an initial demonstration of laboratory capability
and the analysis of spiked samples as a continuing check on performance. The laboratory is
required to maintain performance records to define the quality of data that is generated.
8.1.1 Before performing any analyses, the analyst must demonstrate the ability to generate
acceptable accuracy and precision with this method. This ability is established as
described in Section 8.2.
8.1.2 In recognition of the rapid advances occurring in chromatography, the analyst is
permitted certain options to improve the separations or lower the cost of measurements.
Each time such modifications to the method are made, the analyst is required to repeat
the procedure in Section 8.2.
8.1.3 The laboratory must spike and analyze a minimum of 10% of all samples to monitor
continuing laboratory performance. This procedure is described in Section 8.4.
8.2 To establish the ability to generate acceptable accuracy and precision, the analyst must perform
the following operations.
8.2.1 Select a representative spike concentration for each compound to be measured. Using
stock standards, prepare a quality control check sample concentrate in methanol such that
175
-------
Method 618
a 4-,zL aliquot of the check sample concentrate in 20 mL of water gives the selected
concentration.
8.2.2 Using a 10- tL syringe, add 4 ,zL of the check sample concentrate to each of a minimum
of four 20-niL aliquots of reagent water. A representative wastewater may be used in
place of the reagent water, but one or more additional aliquots must be analyzed to
determine background levels, and the spike level must exceed twice the background level
for the test to be valid. Analyze the aliquots according to the method beginning in
Section 10.
8.2.3 Calculate the average percent recovery (R), and the standard deviation of the percent
recovery (s), for the results. Wastewater background corrections must be made before
R and s calculations are performed.
8.2.4 Using the appropriate data from Table 2, determine the recovery and single-operator
precision expected for the method, and compare these results to the values measured in
Section 8.2.3. If the data are not comparable, review potential problem areas and repeat
the test.
8.3 The analyst must calculate method performance criteria and define the performance of the
laboratory for each spike concentration and compound being measured.
8.3.1 Calculate upper and lower control limits for method performance as follows:
Upper Control Limit (UCL) R + 3s
Lower Control Limit (LCL) = R - 3s
where R and s are calculated as in Section 8.2.3. The UCL and LCL can be used to
construct control charts 5 that are useful in observing trends in performance.
8.3.2 The laboratory must develop and maintain separate accuracy statements of laboratory
performance for wastewater samples. An accuracy statement for the method is defined
as R ± a. The accuracy statement should be developed by the analysis of four aliquots
of wastewater as described in Section 8.2.2, followed by the calculation of R and s.
Alternatively, the analyst may use four wastewater data points gathered through the
requirement for continuing quality control in Section 8.4. The accuracy statements
should be updated regularly. 5
8.4 The laboratory is required to collect in duplicate a portion of their samples to monitor spike
recoveries. The frequency of spiked sample analysis must be at least 10% of all samples or one
sample per month, whichever is greater. One aliquot of the sample must be spiked and analyzed
as described in Section 8.2. If the recovery for a particular compound does not fall within the
control limits for method performance, the results reported for that compound in all samples
processed as part of the same set must be qualified as described in Section 13.3. The laboratory
should monitor the frequency of data so qualified to ensure that it remains at or below 5%.
8.5 Before processing any samples, the analyst should demonstrate through the analysis of a 20-mL
aliquot of reagent water that all glassware and reagent interferences are under control. Each time
a set of samples is extracted or there is a change in reagents, a laboratory reagent blank should
be processed as a safeguard against laboratory contamination.
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Method 618
8.6 It is recommended that the laboratory adopt additional quality assurance practices for use with this
method. The specific practices that are most productive depend upon the needs of the laboratory
and the nature of the samples. Field duplicates may be analyzed to monitor the precision of the
sampling technique. When doubt exists over the identification of a peak on the chromatogram,
confirmatory techniques such as gas chromatography with a dissimilar column, specific element
detector, or mass spectrometer must be used. Whenever possible, the laboratory should perform
analysis of quality control materials and participate in relevant performance evaluation studies.
9. SAMPLE COLLECTiON, PRESERVATiON, AND HANDLING
9.1 Grab samples must be collected in glass containers having a total volume of at least 25 mL. Fill
the sample bottle just to overflowing in such a manner that no air bubbles pass through the sample
as the bottle is being filled. Seal the bottle so that no air bubbles are entrapped in it. Store the
sample in an inverted position and maintain the hermetic seal on the sample bottle until the time
of analysis.
9.2 The samples must be iced or refrigerated at 4°C from the time of collection until extraction.
10. SAMPLE EXTRAC71ON
10.1 Measure 20 mL of sample by pouring the sample into a 40-mL centrifuge tube equipped with a
PTFE-lined screw-cap to. a predetermined 20-mL mark. Adjust pH of sample to 6 to 8 by
addition of 6N sodium hydroxide or 6N sulfuric acid. Measure 4.0 mL of extraction solvent with
a 4-mL graduated pipette and add to the centrifuge tube.
10.2 Shake the tube vigorously for 1 minute. Allow the layers to separate for at least 10 minutes.
Centrifuge, if necessary, to facilitate phase separation.
10.3 Withdraw an aliquot of the solvent layer and proceed with gas chromatographic analysis.
11. CLEANUP AND SEPARA TION
11.1 Cleanup procedures are not generally necessary. If particular circumstances demand the use of
a cleanup procedure, the analyst must determine the elution profile and demonstrate that the
recovery of each compound of interest is no less than 85%.
12. GAS CHROMATOGRAPHY
12.1 Table I summarizes the recommended operating conditions for the gas chromatograph. Included
in this table are estimated retention times and method detection limits that can be achieved by this
method. Examples of the separations achieved by Columns 1 and 2 are shown in Figures 1 and 2,
respectively. Other packed columns, chromatographic conditions, or detectors may be used if the
requirements of Section 8.2 are met. Capillary (open-tubular) columns may also be used if the
relative standard deviations of responses for replicate injections are demonstrated to be less
than 6% and the requirements of Section 8.2 are met.
12.2 Calibrate the system daily as described in Section 7.
12.3 If the internal standard approach is being used, the analyst must not add the internal standard to
sample extracts until immediately before injection into the instrument. Mix thoroughly.
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Method 618
12.4 Inject 1 to 5 1 iL of the sample extract using the solvent-flush technique.’ Record the volume
injected to the nearest 005 L, and the resulting peak size in area or peak height units. An
automated system that consistently injects a constant volume of extract may also be used.
12.5 The width of the retention-time window used to make identifications should be based upon
measurements of actual retention-time variations of standards over the course of a day. Three
times the standard deviation of a retention time can be used to calculate a suggested window size
for a compound. However, the experience of the analyst should weigh heavily in the
interpretation of chromatograms.
12.6 If the response for the peak exceeds the working range of the system, dilute the extract and
reanalyze.
12.7 If the measurement of the peak response is prevented by the presence of interferences, further
cleanup is required.
13. CALCULATiONS
13.1 Determine the concentration of individual compounds in the sample.
13.1.1 If the external standard calibration procedure is used, calculate the amount of material
injected from the peak response using the calibration curve or calibration factor in
Section 7.22. The concentration in the sample can be calculated as follows:
Equation 2
Concentration, gIL = _____
where
A = Amount of material injected, in ng
V 1 = Volume of extract ugected, us iL
V 1 = Volume of total extract, in 1 &L
= Volume of water extracted, In mL
13.1.2 If the internal standard calibration procedure is used, calculate the concentration in the
sample using the response factor (RF) determined in Section 7.3.2 as follows:
Equation 3
Concentration, pgIL = (Afr)(RF)(V,)
where
A, = Response for parwneter to be measured
= Response for the Internal standard
1, = Amount of internal standard added to each extract, in tg
V. = Volume of water extracted, in L
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Method 618
13.2 Report results in micrograms per liter without correction for recovery data. When duplicate and
spiked samples are analyzed, report all data obtained with the sample results.
13.3 For samples processed as part of a set where the laboratory spiked sample recovery falls outside
of the control limits in Section 8.3, data for the affected compounds must be labeled as suspect.
14. GC/MS COP /FIRMA liON
14.1 It is recommended that GC/MS techniques be judiciously employed to support qualitative
identifications made with this method. The mass spectrometer should be capable of scanning the
mass range from 35 amu to a mass 50 amu above the molecular weight of the compound. The
instrument must be capable of scanning the mass range at a rate to produce at least 5 scans per
peak but not to exceed 7 seconds per scan utilizing a 70 V (nominal) electron energy in the
electron impact ionization mode. A GC to MS interface constructed of all glass or glass-lined
materials is recommended. A computer system should be interfaced to the mass spectrometer that
allows the continuous acquisition and storage on machine-readable media of all mass spectra
obtained throughout the duration of the chromatographic program.
14.2 Gas chromatographic columns and conditions should be selected for optimum separation and
performance. The conditions selected must be compatible with standard GC/MS operating
practices. Chromatographic tailing factors of less than 5.0 must be achieved. 9
14.3 At the beginning of each day that confirmatory analyses are to be performed, the GCIMS system
must be checked to see that all decafluorotriphenyl phosphine (DFTPP) performance criteria are
achieved. 7
14.4 To confirm an identification of a compound, the background-corrected mass spectrum of the
compound must be obtained from the sample extract and compared with a mass spectrum from
a stock or calibration standard analyzed under the same chromatographic conditions. It is
recommended that at least 25 ng of material be injected into the GC/MS. The criteria below must
be met for qualitative confirmation.
14.4.1 All ions that are present above 10% relative abundance in the mass spectrum of the
standard must be present in the mass spectrum of the sample with agreement to ±10%.
For example, if the relative abundance of an ion is 30% in the mass spectrum of the
standard, the allowable limits for the relative abundance of that ion in the mass spectrum
for the sample would be 20 to 40%.
14.4.2 The retention time of the compound in the sample must be within 6 seconds of the same
compound in the standard solution.
14.4.3 Compounds that have very similar mass spectra can be explicitly identified by GC/MS
only on the basis of retention time data.
14.5 Where available, chemical ionization mass spectra may be employed to aid in the qualitative
identification process.
14.6 Should these MS procedures fail to provide satisfactory results, additional steps may be taken
before reanalysis. These may include the use of alternate packed or capillary GC columns or
additional cleanup (Section 11).
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Method 618
15. METHOD PERFORMANCE
15.1 The method detection limit (MDL) is defined as the minimum concentration of a substance that
can be measured and reported with 99% confidence that the value is above zero.’° The MDL
concentrations listed in Table I were obtained using reagent water.’ Similar results were achieved
using representative wastewaters.
15.2 This method has been tested for linearity of recovery from spiked reagent water and has been
demonstrated to be applicable over the concentration range from 10 x MDL to 1000 x MDL.
15.3 In a single laboratory, Batteile Columbus Laboratories, using spiked wastewater samples, the
average recoveries presented in Table 2 were obtained. Seven replicates each of two different
wastewaters were spiked and analyzed. The relative standard deviations of the percent recovery
of these measurements are also included in Table 2.
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Method 618
References
1. ASTM Annual Book of Standards, Part 31, D3694, “Standard Practice for Preparation of Sample
Containers and for Preservation,’ American Society for Testing and Materials, Philadelphia, PA,
p. 679, 1980.
2. “Carcinogens - Working with Carcinogens,” Department of Health, Education, and Welfare,
Public Health Service, Center for Disease Control, National Institute for Occupational Safety and
Health, Publication No. 77-206, Aug. 1977.
3. “OSHA Safety and Health Standards, General Industry,” (29 CFR 1910), Occupational Safety and
Health Administration, OSHA 2206 (Revised, January 1976).
4. “Safety in Academic Chemistry Laboratories,” American Chemical Society Publication,
Committee on Chemical Safety, 3rd Edition, 1979.
5. “Handbook for Analytical Quality Control in Water and Wastewater Laboratories,”
EPA-600/4-79-019, U.S. Environmental Protection Agency, Environmental Monitoring and
Support Laboratory - Cincinnati, Ohio 45268, March 1979,.
6. Burke, J.A., “Gas Chromatography for Pesticide Residue Analysis; Some Practical Aspects,”
Journal of the Association of Official Analytical Chemists, 48 1037 (1965).
7. Eichelberger, J.W., Harris, L. E., and Budde, W. L., “Reference Compound to Calibrate Ion
Abundance Measurement in Gas Chromatography-Mass Spectrometry”, Analytical Chemist,y, 47,
995 (1975).
8. “Development of Methods for Pesticides in Wastewaters,” Report from Battelle’s Columbus
Laboratories for EPA Contract 68-03-2956 (in preparation).
9. McNair, H.M., and Bonelli, E.J., Basic Chromatography, Consolidated Printing, Berkeley,
California, 52 (1969).
10. Glaser, l.A. et a!., “Trace Analysis for Wastewaters,” Environmental Science and Technology,
15, 1426 (1981).
181
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Method 678
Table 1. Chromatographic Conditions and Estimated Method Detection Limits
Retention Time ( Mioutes ) Method Detection Limits
Parameter Column 1 Column 2 (p/U
Chloropicrin 5.60 2.03 0.8
Ethylene Dibromide 9.90 3.15 0.2
Column 1 Conditions: Carbopak B (60/80 mesh) coated with 1% SP-1000 packed in a 1.8 m long by
2 mm ID glass column with nitrogen carrier gas at a flow rate of 30 mL/minutes. Column temperature,
isothermal at 135°C. An electron capture detector was used with this column to determine the MDL.
Column 2 Conditions: Gas Chrom Q (100/120 mesh) coated iwth 30% OV-17 packed in a 1.8 m long
by 2 mm ID glass column with helium carrier gas at a flow rate of 25 mL/minutes. Column temperature,
isothermal at 95°C.
Table 2. Single-Laboratory Accuracy and Precision (a)
Spike Level Mean Standard
Sample 8.ckg,uund pg/U Recovery Deviation No. of
Parameter Type(b) pgIL(c) (%) (%J Replicates
Chioropicrin 1 NJ) 5 98 12 7
2 ND 50 98 3.3 7
Ethylene 1 ND 5 69 6.9 7
Dlbronüde 2 ND 50 108 4.8 7
(a) Column I conditions were used.
(b) 1 = Low background relevant industrial effluent
2 = High background relevant industrial effluent
(C) ND = Not detected
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MethOd 618
Figure 1. GC-ECD Chromatogram of 200 ng Chloropicnn and Ethylene
Dibromide in Cyclohexane (Column 1).
Ethylene
I ChIOrOplCnn Dibromide
2.2 3.3 4.4
Retention Time (minutes)
A52-002-20
9.9
11.0
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Mthod 618
C—
/ Ethylene Dbionide
I i f t- I I I I I
0 2.0 4.0 6.0 8.0 10.0 12.0
R fflen m )
Figure 2. GC-ECD Chromatogram of 400 ng Chloropicnn and Ethylene
Dibromide in Cyclohexane (Column 2).
184
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Method 619
The Determination of
Triazine Pesticides in
Municipal and Industrial
Waste water
-------
Method 619
The Determination of Triazine Pesticides in Municipal and
Industrial Waste water
ScoPE AND APPLICATION
1.1 This method covers the determination of certain triazine pesticides. The following parameters can
be determined by this method:
Parameter STORET No. CAS No.
Ametryn — 834-12-8
Atraton — 1610-17-9
Atrazine 39033 19 12-24-9
Prometon 39056 1610-18-0
Prometryn 39057 7287-19-6
Propazine 39024 139-40-2
Seebumeton — 26259-45-0
Simetryn 39054 1014-70-6
Simazine 39055 122-34-9
Terbuthylazine — 5915-41-3
Terbutryn 86-50-0
1.2 This is a gas chromatographic (GC) method applicable to the determination of the compounds
listed above in industrial and municipal discharges as provided under 40 CFR 136.1. Any
modification of this method beyond those expressly permitted shall be considered a major
modification subject to application and approval of alternative test procedures under 40 CFR 136.4
and 136.5.
1.3 The estimated method detection limit (MDL, defined in Section 15) for each parameter is listed
in Table 1. The MDL for a specific wastewater may differ from those listed, depending upon the
nature of interferences in the sample matrix.
1.4 The sample extraction and concentration steps in this method are essentially the same as several
others in 600-series methods. Thus, a single sample may be extracted to measure the parameters
included in the scope of each of these methods. When cleanup is required, the concentration
levels must be high enough to permit selecting aliquots, as necessary, in order to apply appropriate
cleanup procedures. Under gas chromatography, the analyst is allowed the latitude to select
chromatographic conditions appropriate for the simultaneous measurement of combinations of
these parameters (see Section 12).
1.5 This method is restricted to use by or under the supervision of analysts experienced in the use of
gas chromatography and in the interpretation of gas chromatograms. Each analyst must
demonstrate the ability to generate acceptable results with this method using the procedure
described in Section 8.2.
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Method 619
1.6 When this method is used to analyze unfamiliar samples for any or all of the compounds above,
compound identifications should be supported by at least one additional qualitative technique.
This method describes analytical conditions for a second gas chromatographic column that can be
used to confirm measurements made with the primary column. Section 14 provides gas
chromatograph/mass spectrometer (GC/MS) criteria appropriate for the qualitative confirmation
of compound identifications.
2. SUMMARY OF METHOD
2.1 A measured volume of sample, approximately 1 L, is extracted with 15% methylene chloride
using a separatory funnel. The methylene chloride extract is dried and exchanged to hexane
during concentration to a volume of 10 mL or less. Gas chromatographic conditions are described
which permit the separation and measurement of the compounds in the extract by gas
chromatography with a thermionic bead detector in the nitrogen mode.
22 Method 619 represents an editorial revision of a previously promulgated U.S. EPA method for
organophosphorus pesticides.’ While complete method validation data is not presented herein, the
method has been in widespread use since its promulgation, and represents the state of the art for
the analysis of such materials.
2.3 This method provides an optional Florisil column cleanup procedure to aid in the elimination or
reduction of interferences Which may be encountered.
3.
tIW I LtI SWIW .J
3.1 Method interferences may be caused by contaminants in solvents, reagents, glassware, and other
sample-processing apparatus that lead to discrete artifacts or elevated baselines in gas
chrom*ograms. All reagents and apparatus must be routinely demonstrated to be free from
interferences under the conditions of the analysis by running laboratory reagent blanks as
described in Section 8.5.
3.1.1
Glassware must be scrupulously cleaned. 4 Clean all glassware as soon as possible after
use by thoroughly rinsing with the last solvent used in it. Follow by washing with hot
water and detergent and thorough rinsing with tap and reagent water. Drain dry, and heat
in an oven or muffle furnace at 400°C for 15 to 30 minutes. Do not heat volumetric
ware. Thermally stable materials, such as PCBs, may not be eliminated by this
treatment. Thorough rinsing with acetone and pesticide-quality hexane may be substituted
for the heating. After drying and cooling, seal and store glassware in a clean
environment to prevent any accumulation of dust or other contaminants. Store inverted
or capped with aluminum foil.
3.1.2 The use of high-purity reagents and solvents helps to minimize interference problems.
Purification of solvents by distillation in all-glass systems may be required.
3.2 Matrix interferences may be caused by cont min2nts that are coextracted from the sample. The
extent of matrix interferences will vary considerably from source to source, depending upon the
n ire and diversity of the industrial complex or municipality sampled. The cleanup procedure
in Section 11 can be used to overcome many of these interferences, but unique samples may
require ndditional cleanup approaches to achieve the MDL listed in Table 1.
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Method 619
4. SAFETY
4.1 The toxicity or carcinogenicity of each reagent used in this method has not been precisely defined;
however, each chemical compound must be treated as a potential health hazard. From this
viewpoint, exposure to these chemicals must be reduced to the lowest possible level by whatever
means available. The laboratory is responsible for maintaining a current awareness file of OSHA
regulations regarding the safe handling of the chemicals specified in this method. A reference file
of material data handling sheets should also be made available to all personnel involved in the
chemical analysis. Additional references to laboratory safety are available and have been
identified 7 for the information of the analyst.
5. APPARA TI/S AND MA TERIALS
5.1 Sampling equipment, for discrete or composite sampling.
5.1.1 Grab-sample bottle: Amber borosilicate or flint glass, 1-L or 1-quart volume, fitted with
screw-caps lined with TFE-fluorocarbon. Aluminum foil may be substituted for TFE if
the sample is not corrosive. If amber bottles are not available, protect samples from
light. The container and cap liner must be washed, rinsed with acetone or methylene
chloride, and dried before use to minimize contamination.
5.1.2 Automatic sampler (optional): Must incorporate glass sample containers for the
collection of a minimum of 250 mL. Sample containers must be kept refrigerated at 4°C
and protected from light during compositing. If the sampler uses a peristaltic pump, a
minimum length of compressible silicone rubber tubing may be used. Before use,
however, the compressible tubing must be thoroughly rinsed with methanol, followed by
repeated rinsings with reagent water to minimize the potential for contamination of the
sample. An integrating flow meter is required to collect flow-proportional composites.
5.2 Glassware. (All specifications are suggested. Catalog numbers are included for illustration only.)
5.2.1 Separatory funnel: 2000-mL, with TFE-fluorocarbon stopcock, ground-glass or TFE
stopper.
5.2.2 Drying column: Chromatographic column 400 mm long by 19 mm ID with coarse-fitted
disc.
5.2.3 Chromatographic column: 400 mm long by 19 mm ID with coarse-fitted disc at bottom
and TFE-fluorocarbon stopcock (Kontes K-420540-0224 or equivalent).
5.2.4 Concentrator tube, Kuderna-Danish: l0-mL, graduated (Kontes K-570050-1025 or
equivalent). Calibration must be checked at the volumes employed in the test. Ground-
glass stopper is used to prevent evaporation of extracts.
5.2.5 Evaporative flask, Kuderna-Danish: 500-mL (Kontes K-570001-0500 or equivalent).
Attach to concentrator tube with springs.
5.2.6 Snyder column, Kuderna-Danish: Three-ball macro (Kontes K-503000-0121 or
equivalent).
5.2.7 Vials: Amber glass, 10- to 15-mL capacity with TFE-fluorocarbon-lined screw-cap.
5.3 Boiling chips: Approximately 10140 mesh. Heat at 400°C for 30 minutes or perform a Soxhiet
extraction with methylene chloride.
189
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Method 619
5.4 Water bath: Heated, with concentric ring cover, capable of temperature control (± 2°C). The
bath shouldbeused inahood.
5.5 Balance: Analytical, capable of accurately weighing to the nearest 0.0001 g.
5.6 Gas chromatograph: Analytical system complete with gas chromatograph suitable for on-column
injection and all required accessories including syringes, analytical columns, gases, detector, and
strip-chart recorder. A data system is recommended for measuring peak areas.
5.6.1 Column 1: 180 cm long by 2 mm ID glass, packed with 5% Carbowax 20M-TPA on
Supelcoport (80/100 mesh) or equivalent This column was used to develop the method
performance statements in Section 15. Alternative columns may be used in accordance
with the provisions described in Section 12.1.
5.6.2 Column 2: 180 cm long by 4 mm 11) glass, packed with 1.0% Carbowax 20M on Gas
Chrom Q (100/120 mesh) or equivalent.
5.6.3 Detector: Thermionic bead in the nitrogen mode. This detector has proven effective in
the analysis of wastewaters for the parameters listed in the scope and was used to develop
the method performance statements in Section 15. Alternative detectors, including a mass
spectrometer, may be used in accordance with the provisions described in Section 12.1.
6. REAGENTS
6.1 Reagent watec Reagent water is defined as a water in which an interferent is not observed at the
method detection limit of each parameter of interest.
6.2 Acetone, hexane, methylene chloride, methanol: Pesticide-quality or equivalent.
6.3 Ethyl ether: Nanograde, redistilled in glass if necessary. Must be free of peroxides as indicated
by EM Quant test strips (available from Scientific Products Co., Cat. No. P1126-8, and other
suppliers). Procedures recommended for removal of peroxides are provided with the test strips.
Alter cleanup, 20 mL ethyl alcohol preservative must be added to each liter of ether.
6.4 Sodium sulfate: ACS, granular, anhydrous. Condition by heating in a shallow tray at 400°C for
a mininmni of 4 hours to remove phthalates and other interfering organic substances.
Alternatively, heat 16 hours at 450 to 500°C in a sh2Ilow tray or perform a Soxhiet extraction with
methylene chloride for 48 hours.
6.5 Florisil: PR grade (60/100 mesh). Purchase activated at 675°C and store in the dark in glass
container with ground-glass stopper or foil-lined screw-cap. Before use, activate each batch at
least 16 hours at 130°C in a foil-covered glass container.
6.6 Stock standard solutions (1.00 pg/FL): Stock standard solutions may be prepared from pure
standard materials or purchased as certified solutions.
6.6.1 Prepare stock standard solutions by accurately weighing approximately 0.0100 g of pure
material. Dissolve the material in Pesticide-quality hexane or other suitable solvent and
dilute to volume in a 10-mL volumetric flask. Larger volumes may be used at the
convenience of the analyst. If compound purity is certified at 96% or greater, the weight
may be used without correction to calculate the concentration of the stock standard.
Commercially prepared stock standards may be used at any concentration if they are
certified by the manufacturer or by an independent source.
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Method 619
6.6.2 Transfer the stock standard solutions into TFE-fluorocarbon-sealed screw-cap vials.
Store at 4°C and protect from light. Frequently check stock standard solutions for signs
of degradation or evaporation, especially just prior to preparing calibration standards
from them.
6.6.3 Stock standard solutions must be replaced after 6 months, or sooner if comparison with
check standards indicates a problem.
7. CAIJBRA TION
7.1 Establish gas chromatographic operating parameters equivalent to those indicated in Table 1. The
gas chromatographic system may be calibrated using either the external standard technique
(Section 7.2) or the internal standard technique (Section 7.3).
7.2 External standard calibration procedure:
7.2.1 For each parameter of interest, prepare calibration standards at a minimum of three
concentration levels by adding accurately measured volumes of one or more stock
standards to a volumetric flask and diluting to volume with hexane or other suitable
solvent. One of the external standards should be representative of a concentration near,
but above, the method detection limit. The other concentrations should correspond to the
range of concentrations expected in the sample concentrates or should define the working
range of the detector.
7.2.2 Using injections of 1 to 5 iL of each calibration standard, tabulate peak height or area
responses against the mass injected. The results can be used to prepare a calibration
curve for each parameter. Alternatively, the ratio of the response to the mass injected,
defined as the calibration factor (CF), may be calculated for each parameter at each
standard concentration. If the relative standard deviation of the calibration factor is less
than 10% over the working range, the average calibration factor can be used in place of
a calibration curve.
7.2.3 The working calibration curve or calibration factor must be verified on each working
shift by the measurement of one or more calibration standards. If the response for any
parameter varies from the predicted response by more than ± 10%, the test must be
repeated using a fresh calibration standard. Alternatively, a new calibration curve or
calibration factor must be prepared for that parameter.
7.3 Internal standard calibration procedure: To use this approach, the analyst must select one or more
internal standards similar in analytical behavior to the compounds of interest. The analyst must
further demonstrate that the measurement of the internal standard is not affected by method or
matrix interferences. Due to these limitations, no internal standard applicable to all samples can
be suggested.
7.3.1 Prepare calibration standards at a minimum of three concentration levels for each
parameter of interest by adding volumes of one or more stock standards to a volumetric
flask. To each calibration standard, add a known constant amount of one or more
internal standards, and dilute to volume with hexane or other suitable solvent. One of
the standards should be representative of a concentration near, but above, the method
detection limit. The other concentrations should correspond to the range of
191
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Method 619
concentrations expected in the sample concentrates, or should define the working range
of the detector.
7.3.2
Using injections of 1 to 5 jiL of each calibration standard, tabulate the peak height or
area responses against the concentration for each compound and internal standard.
Calculate response factors (RF) for each compound as follows:
Equation 1
= ( A,)(C )
(A,)(C,)
n*ere
A, Response for the parameter to be measured
4, = Response fbr the Internal standard
Cfr = Concentration of the internal standard, in pg/L
C, = Concentration of the parameter to be measured, In pgIL
If the RF value over the working range is constant, less than 10% relative standard
deviation, the RF can be assumed to be invariant and the average RF may be used for
calculations. Alternatively, the results may be used to plot a calibration curve of
response ratios, AJA 1 against RF.
7.3.3 The working calibration curve or RF must be verified on each working shift by the
measurement of one or more calibration standards. If the response for any parameter
varies from the predicted response by more than ± 10%, the test must be repeated using
a fresh calibration standard. Alternatively, a new calibration curve must be prepared for
that compound.
7.4 The cleanup procedure in Section 11 utilizes Florisil chromatography. Florisil from different
batches or sources may vary in adsorptive capacity. To standardize the amount of Florisil which
is used, the use of the lauric acid value is suggested. This procedure’ determines the adsorption
from he ne solution of lauric acid, in milligrams, per gram of Florisil. The amount of Florisil
to be used for each column is calculated by dividing this factor into 110 and multiplying by 20 g.
7.5 Before using any cleanup procedure, the analyst must process a series of calibration standards
through the procedure to validate elution patterns and the absence of interference from the
reagesis.
8. QuAlm’ CoNTRoL
8.1 Each laboratory using this method is required to operate a formal quality control program. The
mininmm requirements of this program consist of an initial demonstration of laboratory capability
and the analysis of spiked samples as a continuing check on performance. The laboratory is
required to m l m performance records to define the quality of data that is generated.
8.1.1 Before performing any analyses, the analyst must demonstrate the ability to generate
acceptable accuracy and precision with this method. This ability is established as
described in Section 8.2.
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Method 619
8.1.2 In recognition of the rapid advances occurring in chromatography, the analyst is
permitted certain options to improve the separations or lower the cost of measurements.
Each time such modifications to the method are made, the analyst is required to repeat
the procedure in Section 8.2.
8.1.3 The laboratory must spike and analyze a minimum of 10% of all samples to monitor
continuing laboratory performance. This procedure is described in Section 8.4.
8.2 To establish the ability to generate acceptable accuracy and precision, the analyst must perform
the following operations.
8.2.1 Select a representative spike concentration for each compound to be measured. Using
stock standards, prepare a quality control check sample concentrate in acetone, 1000
times more concentrated than the selected concentrations.
8.2.2 Using a pipette, add 1.00 mL of the check sample concentrate to each of a minimum of
four 1000-mL aliquots of reagent water. A representative wastewater may be used in
place of the reagent water, but one or more additional aliquots must be analyzed to
determine background levels, and the spike level must exceed twice the background level
for the test to be valid. Analyze the aliquots according to the method beginning in
Section 10.
8.2.3 Calculate the average percent recovery (R), and the standard deviation of the percent
recovery (s), for the results. Wastewater background corrections must be made before
R and s calculations are performed.
8.2.4 Using the data from Table 2, estimate the recovery and single-operator precision expected
for the method, and compare these results to the values calculated in Section 8.2.3. If
the data are not comparable, review potential problem areas and repeat the test.
8.3 The analyst must calculate method performance criteria and define the performance of the
laboratory for each spike concentration and parameter being measured.
8.3.1 Calculate upper and lower control limits for method performance as follows:
Upper Control Limit (UCL) = R + 3s
Lower Control Limit (1CL) = R - 3s
where R and s are calculated as in Section 8.2.3. The UCL and LCL can be used to
construct control charts 9 that are useftil in observing trends in performance.
8.3.2 The laboratory must develop and maintain separate accuracy statements of laboratory
performance for wastewater samples. An accuracy statement for the method is defined
as R ± s. The accuracy statement should be developed by the analysis of four aliquots
of wastewater as described in Section 8.2.2, followed by the calculation of R and s.
Alternatively, the analyst may use four wastewater data points gathered through the
requirement for continuing quality control in Section 8.4. The accuracy statements
should be updated regularly. 9
8.4 The laboratory is required to collect in duplicate a portion of their samples to monitor spike
recoveries. The frequency of spiked sample analysis must be at least 10% of all samples or one
spiked sample per month, whichever is greater. One aliquot of the sample must be spiked and
193
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Method 619
analyzed as described in Section 8.2. If the recovery for a particular parameter does not fall
Within the control limits for method performance, the results reported for that parameter in all
samples processed as part of the same set must be qualified as described in Section 13.3. The
laboratory should monitor the frequency of data so qualified to ensure that it remains at or
below 5%.
8,5 Before processing any samples, the analyst must demonstrate through the analysis of a 1-L aliquot
of reagent water that all glassware and reagent interferences are under control. Each time a set
of samples is extracted or there is a change in reagents, a laboratory reagent blank must be
processed as a safeguard against laboratory contamination.
8.6 It is recommended that the laboratory adopt additional quality assurance practices for use with this
method. The specific practices that are most productive depend upon the needs of the laboratory
and the nature of the samples. Field duplicates may be analyzed to monitor the precision of the
sampling technique. When doubt exists over the identification of a peak on the chromatogram,
confirmatory techniques such as gas chromatography with a dissimilar column, specific element
detector, or mass spectrometer must be used. Whenever possible, the laboratory should perform
analysis of quality control materials and participate in relevant performance evaluation studies.
9. SAMPLE COLLECTION, PRESERVATION, AND HANDLING
9.1 Grab samples must e collected in glass containers. Conventional sampling practices 1 ° should be
followed; however, the bottle must not be prerinsed with sample before collection. Composite
samples should be collected in refrigerated glass containers in accordance with the requirements
of the program. Automatic sampling equipment must be as free as possible of plastic and other
potential sources of contamination.
9.2 The samples must be iced or refrigerated at 4°C from the time of collection until extraction.
9.3 All samples must be extracted within 7 days and completely analyzed within 40 days of extraction.
10. SAMPLE EXTRACTiON
10.1 Mark the water meniscus on the side of the sample bottle for later determination of sample
volume. Pour the entire sample into a 2-L separatory funnel.
10.2 Add 60 mL methylene chloride to the sample bottle, seal, and shake 30 seconds to rinse the inner
walls. Transfer the solvent to the separatory funnel and extract the sample by shaking the funnel
for 2 miimties with periodic v ting to release excess pressure. Allow the organic layer to separate
from the water phase for a minin im of 10 minutes. If the emulsion interface between layers is
more than one-third the volume of the solvent layer, the analyst must employ mechanical
techniques to complete the phase separation. The optimum technique depends upon the sample,
but may include stirring, filtration of the emulsion through glass wool, centrifligation, or other
physical methods. Collect the niethylene chloride extract in a 250-mL Erlenmeyer flask.
10.3 Add a second 60-mL volume of methylene chloride to the sample bottle and repeat the extraction
procedure a second time, combinini the extracts in the Erlenmeyer flask. Perform a third
extraction in the same manner .
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Method 619
10.4 Assemble a Kuderna-Danish (K-D) concentrator by attaching a 10-mL concentrator tube to a
500-mL evaporative flask. Other concentration devices or techniques may be used in place of the
K-D if the requirements of Section 8.2 are met.
10.5 Pour the combined extract through a drying column containing about 10 cm of anhydrous sodium
sulfate, and collect the extract in the K-D concentrator. Rinse the Erlenmeyer flask and column
with 20 to 30 mL of methylene chloride to complete the quantitative transfer.
10.6 Add one or two clean boiling chips to the evaporative flask and attach a three-ball Snyder column.
Prewet the Snyder column by adding about 1 mL methylene chloride to the top. Place the K-D
apparatus on a hot water bath, 60 to 65°C, so that the concentrator tube is partially immersed in
the hot water, and the entire lower rounded surface of the flask is bathed with hot vapor. Adjust
the vertical position of the apparatus and the water temperature as required to complete the
concentration in 15 to 20 minutes. At the proper rate of distillation, the balls of the column will
actively chatter but the chambers will not flood with condensed solvent. When the apparent
volume of liquid reaches 1 mL, remove the K-D apparatus and allow it to drain and cool for at
least 10 minutes.
10.7 Increase the temperature of the hot water bath to about 80°C. Momentarily remove the Snyder
column, add 50 mL of hexane and a new boiling chip, and reattach the Snyder column. Pour
about I mL of hexane into the top of the Snyder column and concentrate the solvent extract as
before. Elapsed time of concentration should be 5 to 10 minutes. When the apparent volume of
liquid reaches 1 mL, remove the K-D apparatus and allow it to drain and cool for at least 10
minutes.
10.8 Remove the Snyder column and rinse the flask and its lower joint into the concentrator tube with
1 to 2 mL of hexane and adjust the volume to 10 mL. A 5-mL syringe is recommended for this
operation.
NO TE: Precipitation of triazines in the hexane may occur if the concentration in the
original sample exceeded 5(X) JLg/L. If this occurs, redissolve the triazines in methylene
chloride and analyze the extract using flame ionization gas chromatography. Stopper the
concentrator tube and store refrigerated if further processing will not be performed
immediately. If the extracts will be stored longer than two days, they should be transferred
to 7FE-fluorocarbon-seaied screw-cap vials. If the sample extract requires no further
cleanup, proceed with gas chromatographic analysis. If the sample requires cleanup,
proceed to Section 11.
10.9 Determine the original sample volume by refilling the sample bottle to the mark and transferring
the water to a 1000-mL graduated cylinder. Record the sample volume to the nearest 5 mL.
11. CLEANUP AND SEPARA TION
11.1 Cleanup procedures may not be necessary for a relatively clean sample matrix. The cleanup
procedure recommended in this method has been used for the analysis of various industrial and
municipal effluents. If particular circumstances demand the use of an alternative cleanup
procedure, the analyst must determine the elution profile and demonstrate that the recovery of
each compound of interest for the cleanup procedure is no less than 85%.
195
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Method 619
11.2 The following Florisil column cleanup procedure has been demonstrated to be applicable to the
nine triazine pesticides listed in Table 3.
11.2.1 Add a weight of Florisil (nominally 20 g) predetermined by calibration (Section 7.4
and 7.5) to a chromatographic column. Settle the Florisil by tapping the column. Add
anhyddiumsfttetothtoPoftheFb0fl 5 lt0f0 ainY to2cmdeep. Add
60 mL of hexane to wet and rinse the sodium sulfate and Florisil. Just prior to exposure
of the sodium sulfate to air, stop the elution of the hexane by closing the stopcock on the
chromatography column. Discard the eluate.
11.2.2 Adjust the sample extract volume to 10 mL with hexane and transfer it from the K-D
concentratortubetotheFlorisilcOlumn. Rinsethetubetwice with lto2mLhexafle,
adding each rinse to the column.
11.2.3 Drain the column until the sodium sulfate layer is nearly exposed. Elute the column with
200 niL of 6% ethyl ether in hexane (V/V) (Fraction 1) using a drip rate of about
5 niL/mm. This fraction may be discarded. Place a 500-niL K -I) flask and clean
concentrator tube under the chromatography column. Elute the column into the flask,
using 200 mL of 15% ethyl ether in hexane (WV) (Fraction 2). Perform a third elutlon
using 200 mL of 50% ethyl ether in hexane (WV) (Fraction 3), and a final elution with
200 niL of 100% ethyl ether (Fraction 4), into separate K-D flasks. The elution patterns
for nine of the pesticides arc shown in Table 3.
11.2.4 Concentrate the eluates by standard K-D techniques (Section 10.6), substituting hexane
for the glassware rinses and using the water bath at about 85°C. Adjust final volume to
10 niL with hexane. Analyze by gas chromatograPhy.
12. Gas CHROMATOGRAPHY
12.1 Table 1 summarizes the recommended operating conditions for the gas cbromatograph. Included
in this table are estimated retention times and method detection limits that can be achieved by this
method. An example of the separation achieved by Column 1 is shown in Figure 1. Other
packed columns, chroinatographiC conditions, or detectors may be used if the requirements of
Section 8.2 are met. Capillary (open-tubular) columns may also be used if the relative standard
deviations of responses for replicate injections are demonstrated to be less than 6% and the
requirements of Section 8.2 are met.
12.2 Calibrate the system daily as described in Section 7.
12.3 If the internal standard approach is being used, add the internal standard to sample extracts
immediately before injection into the instrument. Mix thoroughly.
12.4 Inject 1 to 5 j L of the sample extract using the solvent-flush technique.” Record the volume
injected to the nearest 0.05 sL, and the resulting peak size in area or peak height units. An
automated system that consistently injects a constant volume of extract may also be used.
12.5 The width of the retention-time window used to make identifications should be based upon
measurements of actual retention-time variations of standards over the course of a day. Three
times the standard deviation of a retention time can be used to calculate a suggested window size
for a compound. However, the experience of the analyst should weigh heavily in the
interpretation of cbromatograms.
196
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Method 619
12.6 If the response for the peak exceeds the working range of the system, dilute the extract and
reanalyze.
12.7 If the measurement of the peak response is prevented by the presence of interferences, further
cleanup is required.
13. CALCULA TIONS
13.1 Determine the concentration of individual compounds in the sample.
13.1.1 If the external standard calibration procedure is used, calculate the amount of material
injected from the peak response using the calibration curve or calibration factor in
Section 7.2.2. The concentration in the sample can be calculated as follows:
Equation 2
( A)(V )
Concentration, ig/L = (V)(V)
where
A Amount of material i,dected, in ng
V = Volume of extract injected, in pL
Vt = Voiwne of total extract, in LL
Vs = Volume of water extracted, in mL
13.1.2 If the internal standard calibration procedure was used, calculate the concentration in the
sample using the response factor (RF) determined in Section 7.3.2 as follows:
Equation 3
Concentration, zg/L = (A SIV)
where
A 2 = Response for parameter to be measured
A. = Response for the internal standard
= Amount of internal standard added to each
extract,
in
g
V 0 = Volume of water extracted, in L
13.2 Report results in micrograms per liter without correction for recovery data. When duplicate and
spiked samples are analyzed, report all data obtained with the sample results.
13.3 For samples processed as part of a set where the laboratory spiked sample recovery falls outside
of the control limits in Section 83, data for the affected parameters must be labeled as suspect.
197
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Method 619
14. GC/MS CONFIRMA liON
14.1 It is recommended that GC/MS techniques be judiciously employed to support qualitative
compound identifications made with this method. The mass spectrometer should be capable of
scanning the mass range from 35 amu to a mass 50 amu above the molecular weight of the
compound. The instrument must be capable of sc2nning the mass range at a rate to produce at
least 5 scans per peak but not to exceed 7 seconds per scan utilizing a 70 V (nominal) electron
energy in the electron impact ionization mode. A GC to MS interface constructed of all glass or
glass-lined materials is recommended. A computer system should be interfaced to the mass
spectrometer that allows the continuous acquisition and storage on machine-readable media of all
m c spectra obtained throughout the duration of the chromatographic program.
14.2 Gas chromatographic colnmn and conditions should be selected for optimum separation and
performance. The conditions selected must be compatible with standard GC/MS operating
practices. Chromatographic tailing factors of less than 5.0 must be achieved.’ 2
14.3 At the beginning of each day that confirmatory analyses are to be performed, the GC/MS system
must be checked to see that all decafluorotriphenyl phosphine (DFTPP) performance criteria are
achieved.n
14.4 To confirm an identification of a compound, the background-corrected mass spectrum of the
compound must be obtained from the sample extract and compared with a mass spectrum from
a stock or calibratiqn standard analyzed under the same chromatographic conditions. It is
recommended that at least 25 ng of material be injected into the GC/MS. The criteria below must
be met for qualitative confirmation.
14.4.1 All ions that are present above 10% relative abundance in the mass spectrum of the
standard must be present in the m2c-c spectrum of the sample with agreement to ±10%.
For example, if the relative abundance of an ion is 30% in the mass spectrum of the
standard, the allowable limits for the relative abundance of that ion in the mass spectrum
for the sample would be 20% to 40%.
14.4.2 The retention time of the compound in the sample must be within 6 seconds of the same
compound in the standard solution.
14.4.3 Compounds that have very similar m c spectra can be explicitly identified by GCIMS
only on the basis of retention time data.
14.5 Where available, chemical ionization spectra may be employed to aid in the qualitative
Wendflcadon process.
14.6 Should these MS procedures fail to provide satisfactory results, additional steps may be taken
before reanalysis. These may include the use of alternate packed or capillary GC column.c or
additional cleanup (Section 11).
15. METHOD PERFORMANCE
15.1 The method detection limit (MDL) is defined as the minimum concentration of a substance that
can be measured and reported with 99% confidence that the value is above zero.’ 4 The MDL
concentrations listed in Table 1 were estimated from the response of the thermionic bead nitrogen
detector to each compound. The estimate is based upon the amount of material required to yield
198
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Method 619
a signal 5 times the GC background noise, assuming a 5-FL injection from a 1O-mL final extract
of a 1-L sample.
15.2 In a single laboratory (either West Cost Technical Services, Inc., or Midwest Research Institute),
using effluents from pesticide manufacturers and publicly owned treatment works (POTW), the
average recoveries presented in Table 2 were obtained after Florisil cleanup.” 2 The standard
deviations of the percent recoveries of these measurements are also included in Table 2.
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Method 619
References
1. “Pesticide Methods Evaluation,” Letter Report #11 for EPA Contract No. 68-03-2697. Available
from U.S. Environmental Protection Agency, Environmental Monitoring and Support Laboratory,
Cincinnati, Ohio 45268.
2. of Analytical Test Procedures for Organic Pollutants in Wastewater-Application to
Pesticides,” EPA Report 600/4-81-017, U.S. Environmental Protection Agency, Cincinnati, Ohio.
PB 82 132507, National Technical Information Service, Springfield, Virginia.
3. “Methods for Benzidine, Chlorinated Organic Compounds, Pentachiorophenol and Pesticides in
Water and Wastewater,” U.S. Environmental Protection Agency, Environmental Monitoring and
Support Laboratory - Cincinnati, Ohio , September 1978.
4. ASTM Annual Book of Standards, Part 31, D3694, “Standard Practice for Preparation of Sample
Containers and for Preservation, “American Society for Testing and Materials, Philadelphia,
Pennsylvania, p. 679, 1980.
5. “Carcinogens - Working with Carcinogens,” Department of Health, Education, and Welfare,
Public Health Servioe, Center for Disease Control, National Institute for Occupational Safety and
Health, Publication No. 77-206, Aug. 1977.
6. “OSHA Safety and Health Standards, General Industry,” (29 CFR 1910), Occupational Safety and
Health Administration, OSHA 2206, (Revised, January 1976).
7. “Safety in Academic Chemistry Laboratories,” American Chemical Society Publication,
Committee on Chemical Safety, 3rd Edition, 1979.
8. ASTM Annual Book of Standards, Part 31, D3086, Appendix X3, “Standardization of Florisil
Column by Weight Adjustment Based on Adsorption of Lauric Acid,” American Society for
Testing and Materials, Philadelphia, Pennsylvania, p 765, 1980.
9• •H k for Analytical Quality Control in Water and Wastewater Laboratories,”
EPA-600/4-79-019, U. S. Environmental Protection Agency, Environmental Monitoring and
Support Laboratory - Cincinnati, Ohio, March 1979.
10. ASTM Annual Book of Standards, Part 31, D3370, “Standard Practice for Sampling Water,”
American Society for Testing and Materials, Philadelphia, Pennsylvania, p. 76, 1980.
11. Burke, J.A., “Gas Chromatography for Pesticide Residue Analysis; Some Practical Aspects,”
Journal of the Association of Official Analytical Qiemlsts, 48, 1037 (1965).
12. McNair, H.M., and Boneili, E.J., Basic Ozrovnatography, Consolidated Printing, Berkeley,
California, p. 52, 1969.
200
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Method 619
References (cont.)
13. Eichelberger, J.W., Harris, L.E., and Budde, W.L. “Reference Compound to Calibrate Ion
Abundance Measurement in Gas Chromatography-Mass Spectrometry,” Analytical Chenzistiy, 47,
995 (1975).
14. Glaser, J.A. et.al, “Trace Analysis for Wastewaters,” Environmental Science & Technology, 15,
1426 (1981).
201
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Method 619
Table 1. Chromatographic Conditions and Method Detection Limits
Patameter Retention Time (Mm.) Method
Detection Limit
Column 1 Column 2
(pg/LI
Prometon 6.9 4.9 0.03
Atraton — 6.3 ND
Propazine 9.2 6.7 0.03
Terbuthylazine 10.2 7.3 0.03
Secbumeton — 83 ND
Atrazine 12.4 9.4 0.05
Prometryn 13.8 10.3 0.06
Terbutryn 15.4 — 0.05
Simazine 16.3 12.7 0.06
Ametryn 17.7 14.0 0.06
Simetryn 23.0 0.07
ND = Not determined
Column 1 conditions: Supelcoport (80/100 mesh) coated with 5% Carbowax 20M-TPA packed in a
1.8 m long by 2 mm ID glass column with helium carrier gas at a flow rate of 30 mL/min. Column
temperature, isothermal at 200°C. A thermiomc bead detector was used with this column to determine
the MDL.
Column 2 conditions: Gas Chrom Q (100/120 mesh) coated with 1.0% Carbowax 20 M packed in a
1.8 m long by 4 mm ID glass column with helium carrier gas at 80 mL/min flow rate. Column
temperature, isothermal at 155°C.
202
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Method 619
Table 2. Single-Laboratory Accuracy and Precision
Parameter Sample Spike Number Mean Standard
Type* (pg/Li of Recovery Deviation
Replicates (%) (%)
Ametryn 3 4,000 2 104
3 2,000 2 118
Atrazine 3 300 2 108
1 1,000 7 177 15.2
Prometon 1 130 7 67 3.9
2 260 7 51 3.0
Prometryn 3 2,000 2 76
3 50 2 110
Propazine 1 516 7 54 6.5
3 15 116
Simatryn 3 30 2 183
3 15 2 182
Simazine 1 115 7 152 24.3
3 10 2 99
Terbuthylazine 3 100 2 114
3 15 2 100
Terbutryn 1 968 7 83 10.0
2 169 7 89 24.0
* Sample Type
1 - Industrial process water
2 - Industrial effluent
3 - 80% Industrial process water/20% industrial effluent
203
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Method 619
Table 3. Florisil Fractionation Patterns
Parameter Percent Recoveiy by Fraction
No.1 No.2 No.3 No.4
Propazine 0 90 10
Terbuthylazine 0 30 70
Atrazine 0 20 80
Ametryn 100
Prometryn 100
Simazine 100
Atraton 100
Secbumeton 100
Prometon 100
Florisil eluate composition by fraction
Fraction 1- 200 mL of 6% ethyl ether in hexane
Fraction 2- 200 mL of 15% ethyl ether in hexane
Fraction 3-200 niL of 50% ethyl ether in hexane
Fraction 4-200 mL of ethyl ether
204
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Method 619
Retention Time (minutes)
,Simetryn
25.0
Figure 1. Gas Chromatogram of Triazine Pesticides on Column 1.
For Conditions, See Table 1.
/ Prometon
,Propazine
,Terbuthy lazine
,Atrazine
Prometryn
Terbutryn
0 5.0 10.0 15.0
20.0
A52-cX .49
205
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Method 620
The Determination of
Diphenylamine in Municipal
and Industrial Waste water
-------
Method 620
The Determination of Diphenylamine in Municipal and
Industrial Waste water
1. SCOPE AND APPLICA TION
1.1 This method covers the determination of diphenylamine, CAS. No. 122-39-4.
1.2 This is a gas chromatographic (GC) method applicable to the determination of diphenylamine in
municipal and industrial discharges.
1.3 The method detection limit (MDL, defined in Section 15) for diphenylamine is listed in Table 1.
The MDL for a specific wastewater may differ from those listed, depending upon the nature of
interferences in the sample matrix.
1.4 The sample extraction and concentration steps in this method are similar to those of other 600-
series methods. Thus, a single sample may be extracted to measure the compounds included in
the scope of the methods. When cleanup is required, the concentration levels must be high
enough to permit selecting aliquots, as necessary, in order to apply appropriate cleanup
procedures.
1.5 This method is restricted to use by or under the supervision of analysts experienced in the use of
gas chromatography and in the interpretation of gas chromatograms. Each analyst must
demonstrate the ability to generate acceptable results with this method using the procedure
described in Section 8.2.
1.6 When this method is used to analyze unfamiliar samples for any or all of the compounds above,
compound identifications should be supported by at least one additional qualitative technique.
This method describes analytical conditions for a second gas chromatographic column that can be
used to confirm measurements made with the primary column. Section 14 provides gas
chromatograph/mass spectrometer (GCIMS) criteria appropriate for the qualitative confirmation
of compound identifications.
2. SUMMARY OF METHOD
2.1 A measured volume of sample, approximately 1 L, is extracted with methylene chloride using a
continuous extractor. The methylene chloride extract is dried and concentrated to 5.0 mL. Gas
chromatographic conditions are described which permit the separation and measurement of the
compounds in the extract by alkali flame detector (AFD) gas chromatography.’
2.2 This method provides an optional silica gel column cleanup procedure to aid in the elimination
of interferences which may be encountered.
3. INTERFERENCES
3.1 Method interferences may be caused by contaminants in solvents, reagents, glassware, and other
sample-processing apparatus that lead to discrete artifacts or elevated baselines in gas
chromatograms. All reagents and apparatus must be routinely demonstrated to be free from
209
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Method 620
interferences under the conditions of the analysis by running laboratory reagent blanks as
described in Section 8.5.
3.1.1 Glassware must be scrupulously cleaned. 2 Clean all glassware as soon as possible after
use by thoroughly rinsing with the last solvent used in it. Follow by washing with hot
water and detergent and thorough rinsing with tap and reagent water. Drain dry, and
heat in an oven or muffle furnace at 400°C for 15 to 30 minutes. Thermally stable
materials, such as PCBs, may not be eliminated by this treatment. Thorough rinsing with
acetone and pesticide-quality hexane may be substituted for the heating. After drying and
cooling, seal and store glassware in a clean environment to prevent any accumulation of
dust or other contaminants. Store inverted or capped with aluminum foil.
3.1.2 The use of high-purity reagents and solvents helps to minimize interference problems.
Purification of solvents by distillation in all-glass systems may be required.
3.2 Matrix interferences may be caused by contaminants that are coextracted from the sample. The
extent of matrix interferences will vary considerably from source to source, depending upon the
natnre and diversity of the industrial complex or municipality being sampled. The cleanup
procedure in Section 11 can be used to overcome many of these interferences, but unique samples
may require additional cleanup approaches to achieve the MDL listed in Table 1.
4. SAFETY
4.1 The toxicity or carciziogenicity of each reagent used in this màthod has not been precisely defined;
however, each chemical compound should be treated as a potential health hazard. From this
viewpoint, exposure to these chemicals must be reduced to the lowest possible level by whatever
means available. The laboratory is responsible for maintaining a current awareness file of OSHA
regulations regarding the safe handling of the chemicals specified in this method. A reference file
of material data handling sheets should also be made available to all personnel involved in the
chemical analysis. Additional references to laboratory safety are available and have been
identified for the information of the analyst.
5. APPARA 77JS AND MA TEPJALS
5.1 Sampling equipment, for discrete or composite sampling.
5.1.1 Grab sample bottle: Amber borosiicate or flint glass, 1-L or 1-quart volume, fitted with
screw-caps lined with flFE/PIFE. Aluminum foil may be substituted for PTFE if the
sample is not corrosive. If amber bottles are not available, protect samples from light.
The container and cap liner must be washed, rinsed with acetone or methylene chloride,
and dried before use to minimi, e contamination.
5.1.2 Automatic sampler (optional): Must incorporate glass sample containers for the
collection of a minimum of 250 mL. Sample containers must be kept refrigerated at 4°C
and protected from light during compositing. If the sampler uses a peristaltic pump, a
minimum length of compressible silicone rubber tubing may be used. Before use,
however, the compressible tubing should be thoroughly rinsed with methanol, followed
by repeated rinsings with reagent water to minimize the potential for contamination of
the sample. An integrating flow meter is required to collect flow-proportional
composites.
210
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Method 620
5.2 Glassware. (All specifications are suggested. Catalog numbers are included for illustration only.)
5.2.1 Continuous extractor: 2000-mL, available from Paxton Woods Glass Shop, Cincinnati,
Ohio, or equivalent.
5.2.2 Drying column: Chromatographic column 400 mm long by 10 mm ID.
5.2.3 Chromatographic column: 400 mm long by 19 mm ID with 250-mL reservoir at the top
and PTFE stopcock (Kontes K-420290 or equivalent).
5.2.4 Concentrator tube, Kuderna-Danish: 25-mL, graduated (Kontes K-570050-1025 or
equivalent). Calibration must be checked at the volumes employed in the test. A
ground-glass stopper is used to prevent evaporation of extracts.
5.2.5 Evaporative flask, Kuderna-Danish: 500-mL (Kontes K570001-0500 or equivalent).
Attach to concentrator tube with springs.
5.2.6 Snyder column, Kuderna-Danish: Three-ball macro (Kontes K503000-0121 or
equivalent).
5.2.7 Snyder column, Kuderna-Danish: Two-ball micro (Kontes 1(569001-0219 or equivalent).
5.2.8 Vials: Amber glass, 10- to 15-mL capacity with PTFE-lined screw-cap.
5.2.9 Volumetric flask: 5-mL with glass stopper.
5.3 Boiling chips: Approximately 10/40 mesh carborundum. Heatto 400°C for 4 hours or extract
in a Soxhiet extractor with methylene chloride.
5.4 Water bath: Heated, capable of temperature control ±2°C. The bath should be used in a hood.
5.5 Balance: Analytical, capable of accurately weighing to the nearest 0.0001 g.
5.6 Gas chromatograph: Analytical system complete with gas chromatograph suitable for on-column
injection and all required accessories including syringes, analytical columns, gases, detector, and
strip-chart recorder. A data system is recommended for measuring peak areas.
5.6.1 Column 1: 180 cm long by 2 mm II) glass, packed with 3% SP2250 on Supelcoport
(100/120 mesh) or equivalent. This column was used to develop the method performance
statements in Section 15. Guidelines for the use of alternative columns are provided in
Section 12.1.
5.6.2 Column 2: 180 cm long by 2 mm ID glass, packed with 3% SP 1000 on Supelcoport
(100/120 mesh) or equivalent.
5.6.3 Detector: Alkali-flame detector (AFD), sometimes referred to as a nitrogen-phosphorous
detector (NPD) or a thermionic-specific detector (TSD). This detector has proven
effective in the analysis of wastewaters for the compounds listed in the scope and was
used to develop the method performance statements in Section 15. Alternative detectors,
including a mass spectrometer, may be used in accordance with the provisions described
in Section 12.1.
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Method 620
6. REAGENTS
6.1 Reagent water: Reagent water is defined as a water in which an mterferent is not observed at the
method detection limit of each parameter of interest.
6.2 Methylene chloride, acetone, methanol, petroleum ether, ethyl ether, toluene (distilled-in-glass
quality or equivalent). Ethyl ether must be free of peroxides as indicated by EM Quant test strips
(available from Scientific Products Co., Catalog No. P1126-8, and other suppliers). Procedures
recommended for removal of peroxides are provided with the test strips.
6.3 Sodium sulfate: ACS, granular, anhydrous; heated in a muffle furnace at 400°C overnight.
6.4 Silica gel: Davison Grade 923, 100-200 mesh; activated by heating for 24 hours at 150°C.
6.5 6N sulfuric acid: Slowly add 16.7 mL of concentrated H 2 S0 4 (94%) to about 50 mL of reagent
water. Dilute to 100 znL with reagent water.
66 6N sodium hydroxide: Dissolve 24.0 grams of sodium hydroxide in 100 mL of reagent water.
6.7 Stock standard solutions (1.00 igJpL): Stock standard solutions can be prepared from pure
standard materials or purchased as certified solutions.
6.7.1 Prepare stock standard solutions by accurately weighing about 0.0100 g of pure material.
Dissolve the material in distilled-in-glass quality methanol and dilute to volume in a
10-mL volumetric flask. Larger volumes can be used at the convenience of the analyst.
If compound purity is certified at 96% or greater, the weight can be used without
correction to calculate the concentration of the stock standard. Commercially prepared
stock standards can be used at any concentration if they are certified by the manufacturer
or by an independent source.
6.7.2 Transfer the stock standard solutions into PTFE-sealed screw-cap bottles. Store at 4°C
and protect from light. Frequently check stock standard solutions for signs of
degradation or evaporation, especially just prior to preparing calibration standards from
them.
6.7.3 Stock standard solutions must be replaced after 6 months, or sooner if comparison with
check standards indicates a problem.
7. CA U8RA lION
7.1 Establish gas chromatographic operating parameters equivalent to those indicated in Table 1. The
gas chromatographic system may be calibrated using either the external standard technique
(Section 7.2) or the internal standard technique (Section 7.3).
7.2 External standard calibration procedure.
7.2.1 For each compound of interest, prepare calibration standards at a minimum of three
concentration levels by adding volumes of one or more stock standards to a volumetric
flask and diluting to volume with toluene. One of the external standards should be at a
concentration near, but above, the method detection limit. The other concentrations
should correspond to the expected range of concentrations found in real samples or
should define the working range of the detector.
7.2.2 Using injections of 2 to 5 pL of each calibration standard, tabulate peak height or area
responses against the mass injected. The results can be used to prepare a calibration
212
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Method 620
curve for each parameter. Alternatively, the ratio of the response to the mass injected,
defined as the calibration factor (CF), can be calculated for each compound at each
standard concentration. If the relative standard deviation of the calibration factor is less
than 10% over the working range, the average calibration factor can be used in place of
a calibration curve.
7.2.3 The working calibration curve or calibration factor must be verified on each working
shift by the measurement of one or more calibration standards. If the response for any
compound varies from the predicted response by more than ± 10%, the test must be
repeated using a fresh calibration standard. Alternatively, a new calibration curve or
calibration factor must be prepared for that parameter.
7.3 Internal standard calibration procedure: To use this approach, the analyst must select one or more
internal standards similar in analytical behavior to the compounds of interest. The analyst must
further demonstrate that the measurement of the internal standard is not affected by method or
matrix interferences. Due to these limitations, no internal standard applicable to all samples can
be suggested, although carbazole has been used successfully in some instances.
7.3.1 Prepare calibration standards at a minimum of three concentration levels for each
parameter of interest by adding volumes of one or more stock standards to a volumetric
flask. To each calibration standard, add a known constant amount of one or more
internal standards, and dilute to volume with toluene. One of the standards should be at
a concentration near, but above, the method detection limit. The other concentrations
should correspond to the expected range of concentrations found in real samples, or
should define the working range of the detector.
7.3.2 Using injections of 2 to 5 L of each calibration standard, tabulate the peak height or
area responses against the concentration for each compound and internal standard. Calcu-
late response factors (RF) for each compound as follows:
Equation 1
( A,)(C , )
(4)(C ,)
where
A, = Response for the parameter to be measured
A = Response for the internal standard
C 1 , = Concentration of the internal standard, in JLg/L
C, = Concentration of the parameter to be measured, in jigiL
If the RF value over the working range is constant, less than 10% relative standard
deviation, the RF can be assumed to be invariant and the average RF can be used for
calculations. Alternatively, the results can be used to plot a calibration curve of response
ratios, AJA 1 . against RF.
7.3.3 The working calibration curve or RF must be verified on each working shift by the
measurement of one or more calibration standards. If the response for any compound
varies from the predicted response by more than ±10%, the test must be repeated using
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Method 620
a fresh calibration standard. Alternatively, a new calibration curve must be prepared for
that compound.
7.4 Before using any cleanup procedure, the analyst must process a series of calibration standards
through the procedure to validate elution patterns and the absence of interferences from the
reagents.
8. QuAun’ CONTROL
8.1 Each laboratory using this method is required to operate a formal quality control program. The
minimum requirements of this program consist of an initial demonstration of laboratory capability
and the analysis of spiked samples as a continuing check on performance. The laboratory is
required to maintain performance records to define the quality of data that is generated.
8.1 .1 Before performing any analyses, the analyst must demonstrate the ability to generate
acceptable accuracy and precision with this method. This ability is established as
described in Section 8.2.
8.1.2 In recognition of the rapid advances occurring in chromatography, the analyst is permited
certain options to improve the separations or lower the cost of measurements. Each time
such modifications to the method are made, the analyst is required to repeat the
procedure in Section 8.2.
8.1.3 The laboratory must spike and analyze a mininmm of 10% of all s2mples to monitor
coudnuhig laboratory performance. This procedure is described in Section 8.4.
8.2 To establish the ability to generate acceptable accuracy and precision, the analyst must perform
the following operations.
8.2.1 Select a repres itt e spike concentration for each compound to be measured. Using
stock standards, prepare a quality control check sample concentrate in methanol, 1000
times more concentrated than the selected concentrations.
8.2.2 Using a pipette, add 1.00 mL of the check sample concentrate to each of a minimum of
four l000-mL aliquots of reagent water. A representative wastewater may be used in
place of the reagent water, but one or more additional aliquots must be analyzed to
determine background levels, and the spike level must exceed twice the background level
fix the test to be valid. Analyze the aliquots according to the method beginning in
Section 10.
8.2.3 Calculate the average percent recovery (R), and the standard deviation of the percent
recovery (s), for the results. Wastewater background corrections must be made before
R and s calculations are performed.
8.2.4 Using the appropriate data from Table 2, determine the recovery and single-operator
precision expected for the method, and compare these results to the values measured in
Section 8.2.3. If the data are not comparable, the analyst must review potential problem
areas and repeat the test.
8.3 The analyst must calculate method performance criteria and define the performance of the
laboratory for each spike concentration and parameter being measured.
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Method 620
8.3.1 Calculate upper and lower control limits for method performance as follows:
Upper Control Limit (UCL) = R + 3s
Lower Control Limit (LCL) = R -3s
where R and s are calculated as in Section 8.2.3. The UCL and LCL can be used to
construct control charts 6 that are useful in observing trends in performance.
8.3.2 The laboratory must develop and maintain separate accuracy statements of laboratory
performance for wastewater samples. An accuracy statement for the method is defined
as R ± s. The accuracy statement should be developed by the analysis of four aliquots
of wastewater as described in Section 8.2.2, followed by the calculation R and s.
Alternatively, the analyst must use four wastewater datapoints gathered through the
requirement for continuing quality control in Section 8.4. The accuracy statements 6
should be updated regularly.
8.4 The laboratory is required to collect in duplicate a portion of their samples to monitor spike
recoveries. The frequency of spiked sample analysis must be at least 10% of all samples or one
sample per month, whichever is greater. One aliquot of the sample must be spiked and analyzed
as described in Section 8.2. If the recovery for a particular compound does not fall within the
control limits for method performance, the results reported for that compound in all samples
processed as part of the same set must be qualified as described in Section 13.3. The laboratory
should monitor the frequency of data so qualified to ensure that it remains at or below 5%.
8.5 Before processing any samples, the analyst should demonstrate through the analysis of a 1-L
aliquot of reagent water that all glassware and reagent interferences are under control. Each time
a set of samples is extracted or there is a change in reagents, a laboratory reagent blank should
be processed as a safeguard against laboratory contamination.
8.6 It is recommended that the laboratory adopt additional quality assurance practices for use with this
method. The specific practices that are most productive depend upon the needs of the laboratory
and the nature of the samples. Field duplicates may be analyzed to monitor the precision of the
sampling technique. When doubt exists over the identification of a peak on the chromatograin,
confirmatory techniques such as gas chromatography with a dissimilar column, specific element
detector, or mass spectrometer must be used. Whenever possible, the laboratory should perform
analysis of standard reference materials and participate in relevant performance evaluation studies.
9. SAMPLES COLLECTiON, PRESERVATION, AND HANDLING
9.1 Grab samples must be collected in glass containers. Conventional sampling practices 7 should be
followed; however, the bottle must not be prerinsed with sample before collection. Composite
samples should be collected in refrigerated glass containers in accordance with the requirements
of the program. Automatic sampling equipment must be as free as possible of plastic and other
potential sources of contamination.
9.2 The samples must be iced or refrigerated at 40°C, from the time of collection until extraction.
9.3 Adjust the pH of the sample to 6 to 8 with 6N sodium hydroxide or 6N sulfuric acid immediately
after sampling.
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Method 620
10. SAMPLE EXThACTON
10.1 Assemble continuous extraction apparatus by placing five to ten carborundum chips into the
500-niL round-bottom flask and attaching to the extraction flask.
10.2 Add 400 mL methylene chloride to the extraction flask. Some methylene chloride should displace
into the round-bottom flask.
10.3 Mark the water meniscus on the side of the sample bottle for later determination of sample
volume. Pour the entire sample into the extraction flask and add sufficient distilled water to fill
the extraction flask (2 L total volume aqueous phase).
10.4 Checkthep}I of thesamplewith wide-rangepH paper and adjustto6to8with 6N sodium
hydroxide or 6N sulfuric acid.
10.5 Connect the stirring apparatus to the extraction flask without the fit touching the sample. Heat
methylene chloride in round-bottom flask to continuous reflux and continue heating for 30 minutes
to 1 hour until Mt is thoroughly wetted with methylene chloride.
10.6 Lower Mt until it just touches the sample and start the stirring apparatus rotating.
10.7 Continuously extract sample for 18 to 24 hours.
10.8 Assemble a Kuderna-Danish (K-D) concentrator by attaching a 1O-mL concentrator tube to a
500-mL evaporative flask. Other concentration devices or techniques may be used in place of the
K-D If the requiremepts of Section 8.2 are met.
10.9 Pour the extract from the round-bottom flask through a drying column containing about 10 cm of
anhydrous sodium sulfate, and collect the extract in the K-D concentrator. Rinse the flask and
column with 20 to 30 ml of methylene chloride to complete the quantitative transfer. Once the
flask rinse has passed through the drying column, rinse the column with 30 to 40 inL of
methylene chloride.
10.lOAdd one to two clean boiling chips to the evaporative flask and attach a three-ball Snyder column.
Prewet the Snyder column by addng about 1 mL niethylene chloride to the top. Place the K-D
apparatus on a hot water bath, 60 In 65°C, so that the concentrator tube is partially immersed in
the hot water, and the entire lower rounded surface of the flask is bathed with hot vapor. Adjust
the vertical position of the apparatus and the water temperature as required to complete the
concentration in 15 to 20 minutes. At the proper rate of distillation, the balls of the column will
actively chatter but the chambers will not flood with condensed solvent. When the apparent
volume of liquid reaches approximately 4 mL, remove the K-D apparatus and allow it to drain
and cool for at least 10 minutes.
10.llRemove the Snyder column and flask and adjust the volume of the extract to 5.0 mL with
methylene chloride. Stopper the concentrator tube and store refrigerated if further processing will
not be performed immediately. If the extract is to be stored longer than two days, transfer the
extract to a screw-capped vial with a YFFE-lined cap. If the sample extract requires no further
cleanup, proceed with solvent exchange to toluene and gas chromatographic analysis as described
in Sections 11.5 aM 12 respectively. If the sample requirescleanup, proceed to Section 11.
10.l2Determine the original sample volume by refilling the sample bottle to the mark and transferring
the wMet to a I000-mL graduated cylinder. Record the sample volume to the nearest 5 mL.
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Method 620
11. CLEANUP AND SEPARA TION
11.1 Cleanup procedures may not be necessary for a relatively clean sample matrix. The cleanup
procedure recommended in this method has been used for the analysis of various clean waters and
industrial effluents. If particular circumstances demand the use of an alternative cleanup
procedure, the analyst must determine the elution profile and demonstrate that the recovery of
each compound of interest is no less than 85%.
11.2 Stir 20 g of silica gel in 100 ml. of acetone and 1.2 mL of reagent water for 30 minutes on a
stirring plate. Transfer the slurry to a chroinatographic column (silica gel may be retained with
a plug of glass wool). Wash the column with 20 mL of methylene chloride and then with 30 mL
of petroleum ether. Use a column flow rate of 2 to 2.5 mL/min throughout the wash and elution
profiles. Add an additional 50 mL of petroleum ether to the head of the column.
11.3 Add the extract from Section 10.11 to the head of the column. Allow the solvent to elute from
the column until the Florisil is almost exposed to the air. Elute the column with 50 mL of 6%
ethyl ether in petroleum ether. Discard this fraction.
11.4 Elute the column with 100 mL of 15% ethyl ether in petroleum ether and collect in a K-D
apparatus.
11.5 Add 2.5 mL of toluene to the fraction. Concentrate the fraction to approximately 4 mL with the
water bath at 75 to 80°C as described in Section 10.10. Transfer the sample to a 5-mL
volumetric flask and dilute to 5 mL with toluene. Proceed with gas chromatographic analysis.
12. GAS CHROMATOGRAPHY
12.1 Table 1 summarizes the recommended operating conditions for the gas chromatograph. Included
in this table are estimated retention times and method detection limits that can be achieved by this
method. An example of the separations achieved by Column 1 and Column 2 are shown in
Figures 1 and 2. Other packed columns, chromatographic conditions, or detectors may be used
if the requirements of Section 8.2 are met. Capillary (open-tubular) columns may also be used
if the relative standard deviations of responses for replicate injections are demonstrated to be less
than 6% and the requirements of Section 8.2 are met.
12.2 Calibrate the gas chromatographic system daily as described in Section 7.
12.3 If an internal standard approach is being used, the analyst must not add the internal standard to
the sample extracts until immediately before injection into the instrument. Mix thoroughly.
12.4 Inject 2 to 5 L of the sample extract using the solvent flush technique. 1 Record the volume
injected to the nearest 0.05 L and the resulting peak sizes in area or peak height units.
12.5 The width of the retention-time window used to make identifications should be based upon
measurements of actual retention-time variations of standards over the course of a day. Three
times the standard deviation of a retention time for a compound can be used to calculate a
suggested window size; however, the experience of the analyst should weigh heavily in the
interpretation of chromatograms.
12.6 If the response for the peak exceeds the working range of the system, dilute the extract and
reanalyze.
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Method 620
12.7 If the measurement of the peak response is prevented by the presence of interferences, further
deanup is required.
13. CALCULATiONS
13.1 Determine the concentration of individual compounds in the sample.
13.1.1 If the external standard calibration procedure is used, calculate the amount of material
injected from the peak response using the calibration curve or calibration factor in Section
7.2.2. The concentration in the sample can be calculated as follows:
Equation 2
Concentration, vigIL =
n ere
A Amount of material Injected, In ng
V = Volume of extract Infected, In 1 cL
1’, = Volume of total extract, In iL
V, = Volume of water extracted, In mL
13.1.2
The internal
sample using
standard calibration procedure was used, calculate the concentration in the
the response factor (RF) determined in Section 7.3.2 as follows:
Equation 3
s :
= (Afr)(RF)(V 0 )
A, = Response Jbr parameter to be measured
= R onseJbr the Internal standard
I, Amount of Internal standard ndded to each extract, In ig
= Volume of water extracted, SaL
13.2 Report results in micrograms per liter without correction for recovery data. When duplicate and
spiked samples are analyzed, report all data obtained with the sample results.
13.3 For samples processed as part of a set where the laboratory spiked sample recovery falls outside
of the control limits in Section 8.3, data for the affected compounds must be labeled as suspect.
14. GC/MS CONRRMA lION
14.1 It is recommended that GC/MS techniques be judiciously employed to support qualitative
identifications made with this method. The mass spectrometer should be capable of scanning the
218
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Method 620
mass range from 35 amu to a mass 50 amu above the molecular weight of the compound. The
instrument must be capable of scanning the mass range at a rate to produce at least 5 scans per
peak but not to exceed 7 seconds per scan utilizing a 70 V (nominal) electron energy in the
electron impact ionization mode. A OC to MS interface constructed of all glass or glass-lined
materials is recommended. When using a fused-silica capillary column, the column outlet should
be threaded through the interface to within a few millimeter of the entrance to the source
ionization chamber. A computer system should be interfaced to the mass spectrometer that allows
the continuous acquisition and storage on machine-readable media of all mass spectra obtained
throughout the duration of the chromatographic program.
14.2 Gas chromatographic columns and conditions should be selected for optimum separation and
performance. The conditions selected must be compatible with standard GC/MS operating
practices. Chromatographic tailing factors of less than 5.0 must be achieved. The calculation of
tailing factors is illustrated in Method 625.
14.3 At the beginning of each day that confirmatory analyses are to be performed, the GCIMS system
must be checked to see that all DFTPP performance criteria are achieved. 9
14.4 To confirm an identification of a compound, the background corrected mass spectrum of the
compound must be obtained from the sample extract and compared with a mass spectrum from
a stock or calibration standard analyzed under the same chromatographic conditions. It is
recommended that at least 25 ng of material be injected into the GC/MS. The criteria below must
be met for qualitative confirmation.
14.4.1
The molecular ion and all other ions that are present above 10% relative abundance in
the mass spectrum of the standard must be present in the mass spectrum of the sample
with agreement to ± 10%. For example, if the relative abundance of an ion is 30% in
the mass spectrum of the standard, the allowable limits for the relative abundance of that
ion in the mass spectrum for the sample would be 20 to 40%.
14.4.2 The retention time of the compound in the sample must be within 30 seconds of the same
compound in the standard solution.
14.4.3 Compounds that have very similar mass spectra can be explicitly identified by GC/MS
only on the basis of retention time data.
14.5 Where available, chemical ionization mass spectra may be employed to aid in the qualitative
identification process.
14.6 Should these MS procedures fail to provide satisfactory results, additional steps may be taken
before reanalysis. These may include the use of alternate packed or capillary GC columns or
additional cleanup (Section 11).
15. METHOD PERFORMANCE
15.1 The method detection limit (MDL) is defined as the minimum concentration of a substance that
can be measured and reported with 99% confidence that the value is above zero. The MDL
concentrations listed in Table 1 were obtained using reagent water. 1 Similar results were achieved
using representative wastewaters.
15.2 This method has been tested for linearity of recovery from spiked reagent water and has been
demonstrated to be applicable over the concentration range from 10 X MDL to 1000 x MDL.
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Method 620
15.3 In a single laboratory, Battelle Columbus Laboratories, using spiked wastewater samples, the
average recoveries presented in Table 2 were obtained. Seven replicates of each of two different
wastewaters were spiked and analyzed. The standard deviation of the percent recovery is also
included in Table 2.1
220
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Method 620
References
1. “Development of Methods for Pesticides in Wastewaters,” EPA Contract Report 68-03-2956 (In
preparation).
2. ASTM Annual Book of Standards, Part 31, D3694, “Standard Practice for Preparation of Sample
Containers and for Preservation,” American Society for Testing and Materials, Philadelphia,
Pennsylvania, p. 679, 1980.
3. “Carcinogens - Working with Carcinogens,” Department of Health, Education, and Welfare,
Public Health Service, Center for Disease Control, National Institute for Occupational Safety and
Health, Publication No. 77-206, August, 1977.
4. “OSHA Safety and Health Standards, General Industry,” (29 CFR 1910), Occupational Safety and
Health Administration, OSHA 2206 (Revised, January 1976).
5. “Safety in Academic Chemistry Laboratories,” American Chemical Society Publications,
Committee on Chemical Safety, 3rd Edition, 1979.
6. “Handbook for Analytical Quality Control in Water and Wastewater Laboratories,”
EPA-600/4-79-019, U.S. Environmental Protection Agency, Environmental Monitoring and
Support Laboratory - Cincinnati, Ohio, March 1979.
7. ASTM Annual Book of Standards, Part 31, D3370, “Standard Practice for Sampling Water,”
American Society for Testing and Materials, Philadelphia, Pennsylvania, p. 76, 1980.
8. Burke, J.A., “Gas Chromatography for Pesticide Residue Analysis; Some Practical Aspects,”
Journal of the Association of Official Analytical Chemists, 48, 1037 (1965).
9. Eichelberger, J.W., Harris, L. E., and Budde, W.L., “Reference Compound to Calibrate Ion
Abundance Measurement in Gas Chromatography - Mass Spectrometry, Analytical ChemLc#y, 47,
995 (1975).
221
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Method 620
Table 1. Chromatographic Conditions and Method Detection Limits
Retention Time ( Minutes ) Method Detection Limit
Parameter Column 1 Column 2 (pg/Li
Diphenylamine 1*1 19.3 1.6
Column 1 conditions: Supelcoport (100/120 mesh) coated with 3% SP-2250 packed in a 1.8 m long by
2 mm ID glass column with helium carrier gas at a flow rate of 30 mL/mrn. Column temperature is held
at 80°C for 4 minutes, programmed from 80°C to 300°C at 8°C/mm and held at 300°C for 4 minutes.
Column 2 conditions: Supelcoport (100/120 mesh) coated with 3% SP-1000 packed in a 1.8 m long by
2 m II) glass column with helium carrier gas at a flow rate of 30 mL/min Column temperature is held
atSO°cfor4mi,prognedfrom8o°Cto2So°Cat8O°C/minandhe ldat25O°Cfor4minuteS.
Table 2. Single-Laboratory Accuracy and Preclsion
Relative
Average Standard SpJke Number
P8cent Deviation Level of Matrix
Parameter Recovery (pg/Li Analyses Type (hi
DiphenyI inme 120 25 5.0 7 1
89 11 50.0 7 1
(a) Column 1 conditions were used.
(b) 1 = Columbus secondary P01W effluent
222
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Method 620
Diphenylamine
cutrtim i i i i i i i i
0 17.0 19.0 21.0 23.0 25.0 27.0 29.0 31.0 33.0 35.0
Retention lime (minutes)
A52-002 .50
Figure 1. GC-AFD Chromatogram of 100 ng of Diphenylamine (Column 1).
223
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R Th *ss)
Figure 2.
GG-FID Chromatogram of 200 ng of Diphenylamine (Column 2).
, 0
I
I • I I I I I I I I I I I I I I I U I
0 19.5 21.3 22.5 24.3 25.5 27.3 28.5 30.3 31.5 33.5
M 41
224
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Method 622
The Determination of
Organ ophosphorus
Pesticides in Municipal and
Industrial Waste water
-------
Method 622
The Determination of Organophosphorus Pesticides in
Municipal and Industrial Waste water
1. SCOPE AND APPLICATION
1.1 This method covers the determination of certain organophosphorus pesticides. The following
parameters can be determined by this method:
Parameter STORET No. CAS No.
Azinphos methyl 39580 86-50-0
Boistar — 35400-43-2
Chiorpyrifos — 292 1-88-2
Chiorpyrifos methyl — 5598-13-0
Coumaphos 81293 56-72-4
Demeton 39560 8065-48-3
Diazinon 39570 333-41-5
Dichlorvos — 62-73-7
Disulfoton 39010 298-04-4
Ethoprop — 13194-48-4
Fensulfothion — 115-90-2
Fenthion 39016 55-38-9
Merphos 39019 150-50-5
Mevinphos — 7786-34-7
Naled — 300-76-5
Parathion methyl 39600 298-00-0
Phorate 39023 298-02-2
Ronnel 39357 299-84-3
Stirofos — 961-11-5
Tokuthion — 34643-464
Trichioronate — 327-98-0
1.2 This is a gas chromatographic (GC) method applicable to the determination of the compounds
listed above in industrial and municipal discharges as provided under 40 CFR 136.1. Any
modification of this method beyond those expressly permitted shall be considered a major
modification subject to application and approval of alternate test procedures under 40 CFR 136.4
and 136.5.
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Method 622
1.3 The estimated method detection limit (MDL, defined in Section 15) for each parameter is listed
in Table 1. The MDL for a specific wastewater may differ from those listed, depending upon the
nature of interferences in the sample matrix.
1.4 The sample extraction and concentration steps in this method are essentially the same as several
others in the 600-series methods. Thus, a single sample may be extracted to measure the
parameters included in the scope of each of these methods. When cleanup is required, the
concentration levels must be high enough to perEnit selecting aliquots, as necessary, in order to
apply appropriate cleanup procedures. Under gas chromatography, the analyst is allowed the
latitude to select chromatographic conditions appropriate for the simultaneous measurement of
combinations of these parameters (see Section 12).
1.5 This method is restricted to use by or under the supervision of analysts experienced in the use of
gas chromatography and in the interpretation of gas chromatograms. Each analyst must
demonstrate the ability to generate acceptable results with this method using the procedure
described in Section 8.2.
1.6 When this method is used to analyze unfamiliar samples for any or all of the compounds above,
compound identifications should be supported by at least one additional qualitative technique.
Section 14 provides gas chromatograph/mass spectrometer (GC/MS) criteria appropriate for the
qualitative confirmation of compound identifications.
2. SUMMARY OF METHOD
2.1 A measured volume of sample, approximately 1 L, is extracted with 15% methylene chloride
using a separatory funnel. The methylene chloride extract is dried and exchanged to hexane
during concentration to a volume of 10 mL or less. Gas chromatographic conditions are described
which permit the separation and measurement of the compounds in the extract by gas
chromatography with a thermionic bead or flame photometric detector in the phosphorus mode. 1
3. INTERFERENCES
3.1 Method interferences may be caused by contaminants in solvents, reagents, glassware, and other
sample-processing apparatus that lead to discrete artifacts or elevated baselines in gas
chromatograms. All reagents and apparatus must be routinely demonstrated to be free from
interferences under the conditions of the analysis by rnnning laboratory reagent blanks as
described in Section 8.5.
3.1.1
Glassware must be scrupulously cleaned. 2 Clean all glassware as soon as possible after
use by thoroughly rinsing with the last solvent used in it. Follow by washing with hot
water and detergent and thorough rinsing with tap and reagent water. Drain dry, and heat
in an oven or muffle furnace at 400°C for 15 to 30 minutes. Do not heat volumetric
ware. Thermally stable materials, such as PCBs, may not be eliminated by this
treatment. Thorough rinsing with acetone and pesticide-quality hexane may be substituted
for the hewing. Alter drying and cooling, seal and store glassware in a clean
environment to prevent any accumulation of dust or other contaminants. Store inverted
or capped with aluminum foil.
3.1.2 The use of high-purity reagents and solvents helps to mininiize interference problems.
Purification of solvents by distillation in all-glass systems may be required.
228
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Method 622
3.2 Matrix interferences may be caused by contaminants that are coextracted from the sample. The
extent of matrix interferences will vary considerably from source to source, depending upon the
nature and diversity of the industrial complex or municipality sampled. Unique samples may
require special cleanup approaches or selective GC detectors to achieve the MDL listed in
Table I. Use of a flame photometric detector in the phosphorus mode will minimize interferences
from materials that do not contain phosphorus. Elemental sulfur, however, may interfere with
the determination of certain organophosphorus pesticides by flame photometric gas
chromatography. A halogen-specific detector (electrolytic conductivity or microcoulometric) is
very selective for the halogen-containing pesticides and has been shown to be effective in the
analysis of wastewater for dichiorvos, naled, and stirofos.
4. SAFETY
4.1 The toxicity or carcinogenicity of each reagent used in this method has not been precisely defined;
however, each chemical compound must be treated as a potential health hazard. From this
viewpoint, exposure to these chemicals must be reduced to the lowest possible level by whatever
means available. The laboratory is responsible for maintaining a current awareness file of OSHA
regulations regarding the safe handling of the chemicals specified in this method. A reference file
of material data handling sheets should also he made available to all personnel involved in the
chemical analysis. Additional references to laboratory safety are available and have been
identified 5 for the information of the analyst.
5. APPARA TUS AND MA TEPJALS
5.1 Sampling equipment, for discrete or composite sampling.
5.1 .1 Grab-sample bottle: Amber borosilicate or flint glass, 1-L or 1-quart volume, fitted with
screw-caps lined with TFE-fluorocarbon. Aluminum foil may be substituted for TFE if
the sample is not corrosive. If amber bottles are not available, protect samples from
light. The container and cap liner must be washed, rinsed with acetone or methylene
chloride, and dried before use to minimize contamination.
5.1.2 Automatic sampler (optional): Must incorporate glass sample containers for the
collection of a minimum of 250 mL. Sample containers must be kept refrigerated at 4°C
and protected from light during compositing. If the sampler uses a peristaltic pump, a
minimum length of compressible silicone rubber tubing may be used. Before use,
however, the compressible tubing must be thoroughly rinsed with methanol, followed by
repeated rinsings with reagent water to minimize the potential for contamination of the
sample. An integrating flow meter is required to collect flow-proportional composites.
5.2 Glassware. (All specifications are suggested. Catalog numbers are included for illustration only.)
5.2.1 Separatory funnel: 2000-mL, with TFE-fluorocarbon stopcock, ground-glass or TFE
stopper.
5.2.2 Drying column: Chromatographic column 400mm long by 19 mm ID with coarse-fitted
disc.
229
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Method 622
5.2.3 Concentrator tube, Kuderna-Danish: 1O-mL, graduated (Kontes K-570050-1025 or
equivalent). Calibration must be checked at the volumes employed in the test. Ground-
glass stopper is used to prevent evaporation of extracts.
5.2.4 Evaporative flask, Kuderna-Danish: 500-mL (Kontes K-570001-0500 or equivalent).
Attach to concentrator tube with springs.
5.2.5 Snyder column, Kuderna-Danish: Three-ball macro (Kontes K-503000-0121 or
equivalent).
5.2.6 Vials: Amber glass, 10- to 15-inL capacity with TFE-fluorocarbon-lined screw-cap.
5.3 Boiling chips: Approximately 10140 mesh. Heat at 400°C for 30 minutes or perform soxhlet
extraction with methylene chloride.
5.4 Water bath: Heated, with concentric ring cover, capable of temperature control (±2°C). The
bath should beused inahood.
5.5 Balance: Analytical, capable of accurately weighing to the nearest 0.0001 g.
5.6 Gas chromatograph: Analytical system complete with gas chromatograph suitable for on-column
injection and all required accessories including syringes, analytical columns, gases, detector and
strip-chart recorder. A data system is recommended for measuring peak areas.
5.6.1 ___
Colnmns: These column . were used to develop the method performance statements in
Section 15. Alternate columns may be used in accordance with the provisions described
in Section 12.1.
5.6.1.1 Column 1: 180 cm long by 2 mm ID glass, packed with 5% SP-2401 on
Supelcopoit (100/120 mesh) or equivalent.
5.6.1.2 Column 2: 180 cm long by 2 mm ID glass, packed with 3% SP-2401 on
Supelcoport (100/120 mesh) or equivalent.
5.6.1.3 Column 3: 50 cm long by ¼ inch OD PTFE, packed with 15% SE-54 on
Gas Chrom Q (80/100 mesh) or equivalent.
5.6.2 Detector: Thermionic bead or flame photometric in the phosphorus mode. These
detectors have proven effective in the analysis of wastewaters for the parameters listed
in the scope and were used to develop the method performance statements in Section 15.
Alternative detectors, including a mass spectrometer, may be used in accordance with the
provisions described in Section 12.1.
6. REAGEN7$
6.1 Reagent water: Reagent water Is defined as a water in which an interferent is net observed at the
method detection limit of each parameter of interest.
6.2 Acetone, hexane, methylene chloride: Pesticide-quality or equivalent.
6.3 Sodium sulfate: ACS, granular, anhydrous. Condition by heating in a shallow tray at 400°C for
a mImn im of 4 hours to remove phthalates sal other interfering organic substances.
Alternatively, heat 16 hours at 450 to 500°C in a shallow tray or perform a Soxhiet extraction
with methylene chloride for 48 hours.
6.5 Stock standard solutions (1.00 JLgl IL): Stock standard solutions may be prepared from pure
standard materials or purchased as certified solutions.
230
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Method 622
6.5.1 Prepare stock standard solutions by accurately weighing approximately 0.0100 g of pure
material. Dissolve the material in pesticide-quality hexane or other suitable solvent and
dilute to volume in a 1O-mL volumetric flask. Larger volumes may be used at the
convenience of the analyst. If compound purity is certified at 96% or greater, the weight
may be used without correction to calculate the concentration of the stock standard.
Commercially-prepared stock standards may be used at any concentration if they are
certified by the manufacturer or by an independent source.
6.5.2 Transfer the stock standard solutions into TFE-fluorocarbon-sealed screw-cap vials.
Store at 4°C and protect from light. Frequently check stock standard solutions for signs
of degradation or evaporation, especially just prior to preparing calibration standards
from them.
6.6.3 Stock standard solutions must be replaced after 6 months, or sooner if comparison with
check standards indicates a problem.
7. CAL /BRA TION
7.1 Establish gas chromatographic operating parameters equivalent to those indicated in Table 1. The
gas chromatographic system may be calibrated using either the external standard technique
(Section 7.2) or the internal standard technique (Section 7.3).
7.2 External standard calibration procedure.
7.2.1 For each parameter of interest, prepare calibration standards at a minimum of three
concentration levels by adding accurately measured volumes of one or more stock
standards to a volumetric flask and diluting to volume with hexane or other suitable
solvent. One of the external standards should be representative of a concentration near,
but above, the method detection limit. The other concentrations should correspond to the
range of concentrations expected iii the sample concentrates or should define the working
range of the detector.
7.2.2 Using injections of I to 5 iL of each calibration standard, tabulate peak height or area
responses against the mass injected. The results can be used to prepare a calibration
curve for each parameter. Alternatively, the ratio of the response to the mass injected,
defined as the calibration factor (CF), may be calculated for each parameter at each
standard concentration. If the relative standard deviation of the calibration factor is less
than 10% over the working range, the average calibration factor can be used in place of
a calibration curve.
7.2.3 The working calibration curve or calibration factor must be verified on each working
shift by the measurement of one or more calibration standards. If the response for any
parameter varies from the predicted response by more than ±10%, the test must be
repeated using a fresh calibration standard. Alternatively, a new calibration curve or
calibration factor must be prepared for that parameter.
7.3 Internal standard calibration procedure: To use this approach, the analyst must select one or more
internal standards similar in analytical behavior to the compounds of interest. The analyst must
further demonstrate that the measurement of the internal standard is not affected by method or
matrix interferences. Due to these limitations, no internal standard applicable to all samples can
be suggested.
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Method 622
7.3.1 Prepare calibration standards at a minimum of three concentration levels for each
parameter of interest by adding volumes of one or more stock standards to a volumetric
flask. To each calibration standard, add a known constant amount of one or more
internal standards, and dilute to volume with hexane or other suitable solvent. One of
the standards should be representative of a concentration near, but above, the method
detection limit. The other concentrations should correspond to the range of
concentrations expected in the sample concentrates, or shouki define the working range
of the detector.
7.3.2 Using injections of 1 to 5 pL of each calibration standard, tabulate the peak height or
area responses against the concentration for each compound and internal standard.
Calculate response factors (RF) for each compound as follows:
EquatIon 1
RF = ( A,)(Cfr )
(A 1 ,)(C,)
where
A, = Response for the parameter to be measured
= kezponse for the internal standard
Cfr = Concentration of the Internal standard, In zgIL
C, = Concentration of the parameter to be measured, in gLgIL
If the RF value over the working range is constant, less than 10% relative standard
deviation, the RF can be assumed to be invariant and the average RF may be used for
calculations. Alternatively, the results may be used to plot a calibration curve of
response ratios, AJ& against RF.
7.3.3 The working calibration curve or RF must be verified on each working shift by the
measurement of one or more calibration standards. If the response for any parameter
varies from the predicted response by more than ± 10%, the test must be repeated using
a fresh calibration standard. Alternatively, a new calibration curve must be prepared for
that compound.
7.4 Before using any cleanup procedure, the analyst must process a series of calibration standards
through the procedure to validate elution patterns and the absence of interference from the
8. QuALITY CONTROL
8.1 Each laboratory using this method is required to operate a formal quality control program. The
mininrum requirements of this program consist of an initial demonstration of laboratory capability
and the analysis of spiked samples as a continuing check on performance. The laboratory is
required to m h*ain performance records to define the quality of data that is generated.
8.1.1 Before performing any analyses, the analyst must demonstrate the ability to generate
acceptable accuracy and precision with this method. This ability is established as
described in Section 8.2.
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Method 622
8.1.2 In recognition of the rapid advances occurring in chromatography, the analyst is
permitted certain options to improve the separations or lower the cost of measurements.
Each time such modifications to the method are made, the analyst is required to repeat
the procedure in Section 8.2.
8.1.3 The laboratory must spike and analyze a minimum of 10% of all samples to monitor
continuing laboratory performance. This procedure is described in Section 8.4.
8.2 To establish the ability to generate acceptable accuracy and precision, the analyst must perform
the following operations.
8.2.1 Select a representative spike concentration for each compound to be measured. Using
stock standards, prepare a quality control check sample concentrate in acetone, 1000
times more concentrated than the selected concentrations.
8.2.2 Using a pipette, add 1.00 mL of the check sample concentrate to each of a minimum of
four 1000-mL aliquots of reagent water. A representative wastewater may be used in
place of the reagent water, but one or more additional aliquots must be analyzed to
determine background levels, and the spike level must exceed twice the background level
for the test to be valid. Analyze the aliquots according to the method beginning in
Section 10.
8.2.3 Calculate the average % recovery (R), and the standard deviation of the % recovery (s),
for the results. Wastewater background corrections must be made before R and s
calculations are performed.
8.2.4 Using the appropriate data from Table 2, determine the recovery and single-operator
precision expected for the method, and compare these results to the values calculated in
Section 8.2.3. If the data are not comparable, review potential problem areas and repeat
the test.
8.3 The analyst must calculate method performance criteria and define the performance of the
laboratory for each spike concentration and parameter being measured.
8.3.1 Calculate upper and lower control limits for method performance as follows:
Upper Control Limit (UCL) = R + 3s
Lower Control Limit (LCL) = R -3s
where R and s are calculated as in Section 8.2.3. The UCL and LCL can be used to
construct control charts 6 that are useful in observing trends in performance.
8.3.2 The laboratory must develop and maintain separate accuracy statements of laboratory
performance for wastewater samples. An accuracy statement for the method is defined
as R ± s. The accuracy statement should be developed by the analysis of four aliquots
of wastewater as described in Section 8.2.2, followed by the calculation of R and s.
Alternatively, the analyst may use four wastewater data points gathered through the
requirement for continuing quality control in Section 8.4. The accuracy statements
should be updated regularly. 6
8.4 The laboratory is required to collect in duplicate a portion of their samples to monitor spike
recoveries. The frequency of spiked sample analysis must be at least 10% of all samples or one
233
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Method 622
spiked sample per month, whichever is greater. One aliquot of the sample must be spiked and
analyzed as described in Section 8.2. If the recovery for a particular parameter does not fall
within the control limits for method performance, the results reported for that parameter in all
samples processed as part of the same set must be qualified as described in Section 1.3. The
laboratory should monitor the frequency of data so qualified to ensure that it remains at or
below 5%.
8.5 Before processing any samples, the analyst must demonstrate through the analysis of a 1-L aliquot
of reagent water that all glassware and reagent interferences are under control. Each time a set
of samples is extracted or there is a change in reagents, a laboratory reagent blank must be
processed as a safeguard against laboratory contamination.
8.6 It is recommended that the laboratory adopt additional quality assurance practices for use with this
method. The specific practices that are most productive depend upon the needs of the laboratory
and the n2lhre of the samples. Field duplicates may be analyzed to monitor the precision of the
sampling technique. When doubt exists over the identification of a peak on the chromatogram,
confirmatory techniques such as gas chromatography with a dissimilar column, specific element
detector, or mass spectrometer must be used. Whenever possible, the laboratory should perform
analysis of quality control materials and participate in relevant performance evaluation studies.
9. SAMPLE COLLECTIoN, PRESERVATiON, AND HANDLING
9.1 Grab samples must be collected in glass containers. Conventional sampling practices 7 should be
followed; however, the bottle must not be prerinsed with sample before collection. Composite
samples should be collected in refrigerated glass containers in accordance with the requirements
of the program. Automatic sampling equipment must be as free as possible of plastic and other
potential sources of contamination.
9.2 The samples imist be iced or refrigerated at 4°C from the time of collection until extraction.
9.3 All samples must be extracted within 7 days and completely analyzed within 40 days of extraction.
10. SAMPLE EXTRACTION
10.1 Mark the water ineniscus on the side of the sample bottle for later determination of sample
volume. Pour the entire sample into a 2-L separatory funnel.
10.2 Add 60 mL methylene chloride to the sample bottle, seal, and shake 30 seconds to rinse the inner
walls. Transfer the solvent to the separatory funnel and extract the sample by shaking the funnel
for 2 minutes with periodic venting to release excess pressure. Allow the organic layer to separate
from the water phase for a minimnm of 10 minutes. If the emulsion interface between layers is
more than one-third the volume of the solvent layer, the analyst must employ mechanical
techniques to complete the phase separation. The optimum technique depends upon the sample,
but may include stirring, filtration of the emulsion through glass wool, centrifugation, or other
physical methods. Collect the methylene chloride extract in a 250-mL Erlenmeyer flask.
10.3 Add a second 60 -mL volume of methylene chloride to the sample bottle and repeat the extraction
procedure a second time, combining the extracts in the Erlenmeyer flask. Perform a third
extraction in the same manner.
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Method 622
10.4 Assemble a Kuderna-Danish (K-D) concentrator by attaching a lO-mL concentrator tube to a
500-mL evaporative flask. Other concentration devices or techniques may be used in place of the
K-D if the requirements of Section 8.2 are met.
10.5 Pour the combined extract through a drying column containing about 10 cm of anhydrous sodium
sulfate, and collect the extract in the K-D concentrator. Rinse the Erlenmeyer flask and column
with 20 to 30 mL of methylene chloride to complete the quantitative transfer.
10.6 Add one or two clean boiling chips to the evaporative flask and attach a three-ball Snyder column.
Prewet the Snyder column by adding about 1 mL methylene chloride to the top. Place the K-D
apparatus on a hot water bath, 60 to 65°C, so that the concentrator tube is partially immersed in
the hot water, and the entire lower rounded surface of the flask is bathed with hot vapor. Adjust
the vertical position of the apparatus and the water temperature as required to complete the
concentration in 15 to 20 minutes. At the proper rate of distillation, the balls of the column will
actively chatter but the chambers will not floOd with condensed solvent. When the apparent
volume of liquid reaches 1 mL, remove the K-D apparatus and allow it to drain and cool for at
least 10 minutes.
10.7 Increase the temperature of the hot water bath to about 80°C. Momentarily remove the Snyder
column, add 50 mL of hexane and a new boiling chip, and reattach the Snyder column. Pour
about I mL of hexane into the top of the Snyder column and concentrate the solvent extract as
before. Elapsed time of concentration should be 5 to 10 minutes. When the apparent volume of
liquid reaches 1 mL, remove the K-D apparatus and allow it to drain and cool for at least 10
minutes.
10.8 Remove the Snyder column and rinse the flask and its lower joint into the concentrator tube with
1 to 2 mL of hexane and adjust the volume to 10 mL. A 5-mL syringe is recommended for this
operation. Stopper the concentrator tube and store refrigerated if further processing will not be
performed immediately. If the extracts will be stored longer than two days, they should be
transferred to TFE-fluorocarbon-sealed screw-cap vials. If the sample extract requires no further
cleanup, proceed with gas chromatographic analysis. If the sample requires cleanup, proceed to
Section 11.
10.9 Determine the original sample volume by refilling the sample bottle to the mark and transferring
the water to a 1000-mL graduated cylinder. Record the sample volume to the nearest 5 mL.
11. CLEANUP AND SEPARA liON
11.1 Cleanup procedures may not be necessary for a relatively clean sample matrix and were not
required for the analysis of the wastewaters reported in Section 15. If particular circumstances
demand the use of a cleanup procedure, the analyst must determine the elution profile and
demonstrate that the recovery of each compound of interest for the cleanup procedure is no less
than 85%.
12. GAs CHROMATOGRAPHY
12.1 Table 1 summarizes the recommended operating conditions for the gas chromatograph. Included
in this table are estimated retention times and method detection limits that can be achieved by this
method. Naled is partially converted to dichiorvos on GC Columns 1 and 2 but not on Column 3.
235
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Method 622
Therefore, if naled is to be measured in the sample, GC analysis for dichlorvos and naled must
be performed using Column 3. Examples of the separations achieved are shown in Figures 1
through 4. Other packed columns, chromatographic conditions, or detectors may be used if the
requirements of Section 8.2 are met. Capillary (open-tubular) columns may also be used if the
relative standard deviations of responses for replicate injections are demonstrated to be less than
6% and the requirements of Section 8.2 are met.
12.2 Calibrate the system daily as described in Section 7.
12.3 If the internal standard approach is being used, add the internal standard to sample extracts
immediately before injection into the instrument. Mix thoroughly.
12.4 Inject I to 5 giL of the sample extract using the solvent-flush technique.’ Record the volume
injected to the nearest 0.05 giL, and the resulting peak size in area or peak height units. An
automated system that consistently injects a constant volume of extract may also be used.
12.5 The width of the retention-time window used to make identifications should be based upon
measurements of actual retention-time variations of standards over the course of a day. Three
times the standard deviation of a retention time can be used to calculate a suggested window size
for a compound. However, the experience of the analyst should weigh heavily in the
interpretation of chromatograms.
12.6 If the response for the peak exceeds the working range of the system, dilute the extract and
reanalyze.
12.7 If the measurement of the peak response is prevented by the presence of interferences, cleanup
is required.
13. CALCULATIONS
13.1 Determine the concentration of individual compounds in the sample.
13.1.1 If the external standard calibration procedure is used, calculate the amount of material
injected from the peak response using the calibration curve or calibration factor in Section
7.2.2. The concentration in the sample can be calculated as follows:
EquatIon 2
Concentration, gigIL (I’XV)
A _Amowltq?materlail4frct.d, lnng
V 1 Volume of extract btjected, In giL
- Volwne (1 t(JtoJ extract, InpL
- Volume of w er extracted, v i niL
13.1.2 If the internal standard calibration procedure was used, calculate the concentration in the
sample using the response factor (RE) determined in Section 7.3.2 as follows:
236
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Method 622
Equation 3
Concentration, g/L = ( A )(l, )
(A 1 ,)(RF)(V 0 )
where
= Response for parameter to be measured
A,, = Response for the internal standard
I , = Amount of internal standard added to each extract, in g
V 0 = Volume of water extracted, in L
13.2 Report results in micrograms per liter without correction for recovery data. When duplicate and
spiked samples are analyzed, report all data obtained with the sample results.
13.3 For samples processed as part of a set where the laboratory spiked sample recovery falls outside
of the control limits in Section 8.3, data for the affected parameters must be labeled as suspect.
14. CC/MS CONFIRMA TION
14.1 It is recommended that GCIMS techniques be judiciously employed to support qualitative
compound identifications made with this method. The mass spectrometer should be capable of
scanning the mass range from 35 amu to a mass 50 amu above the molecular weight of the
compound. The instrument must be capable of scanning the mass range at a rate to produce at
least 5 scans per peak but not to exceed 7 seconds per scan utilizing a 70 V (nominal) electron
energy in the electron impact ionization mode. A GC to MS interface constructed of all glass or
glass-lined materials is recommended. A computer system should be interfaced to the mass
spectrometer that allows the continuous acquisition and storage on machine-readable media of all
mass spectra obtained throughout the duration of the chromatographic program.
14.2 Gas chromatographic columns and conditions should be selected for optimum separation and
performance. The conditions selected must be compatible with standard GC/MS operating
practices. Chromatographic tailing factors of less than 5.0 must be achieved. 9
14.3 At the beginning of each day that confirmatory analyses are to be performed, the GC/MS system
must be checked to see that all decafluorotriphenyl phosphine (DFTPP) performance criteria are
achieved. 10
14.4 To confirm an identification of a compound, the background-corrected mass spectrum of the
compound must be obtained from the sample extract and compared with a mass spectrum from
a stock or calibration standard analyzed under the same chromatographic conditions. It is
recommended that at least 25 ng of material be injected into the GCIMS. The criteria below must
be met for qualitative confirmation.
14.4.1 All ions that are present above 10% relative abundance in the mass spectrum of the
standard must be present in the mass spectrum of the sample with agreement to ±10%.
For example, if the relative abundance of an ion is 30% in the mass spectrum of the
standard, the allowable limits for the relative abundance of that ion in the mass spectrum
for the sample would be 20% to 40%.
237
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Method 622
14.4.2 The retention time of the compound in the sample must be within 6 seconds of the same
compound in the standard solution.
14.4.3 Compounds that have very similar mass spectra can be explicitly identified by GCIMS
only on the basis of retention time data.
14.5 Where available, chemical ionization mass spectra may be employed to aid in the qualitative
identification process.
14.6 Should these MS procedures fail to provide satisfactory results, additional steps may be taken
before reanalysis. These may include the use of alternate packed or capillary (3C columns or
additional cleanup (Section 11).
15. METHoD PERFORMANCE
15.1 The m hod detection limit (MDL) is defined as the minimum concentration of a substance that
can be measured and reported with 99% confidence that the value is above zero.’ 1 The MDL
concentrations listed in Table 1 were estimated from the response of the detector to each
compound. The estimate is based upon the amount of material required to yield a signal 5 times
the GC background noise, assuming a 5-pL injection from a 1O-mL final extract of a 1-L sample.
15.2 In a single Iabor tory (West Cost Technical Services, Inc.), using effluents from pesticide
manufacturers and publicly-owned treatment works (PC)TW), the average recoveries presented in
Table 2 were obtained. 1 The standard deviations of the percent recoveries of these measurements
are also included in Table 2.
238
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Method 622
References
1. “Pesticide Methods Evaluation,” Letter Reports #6, 12A, and 14 for EPA Contract No. 68-03-
2697. Available from U.S. Environmental Protection Agency, Environmental Monitoring and
Support Laboratory, Cincinnati, Ohio.
2. ASTM Annual Book of Standards, Part 31, D3694, “Standard Practice for Preparation of Sample
Containers and for Preservation, “ American Society for Tasting and Materials, Philadelphia,
Pennsylvania, p. 679, 1980.
3. “Carcinogens: Working with Carcinogens,” Department of Health, Education, and Welfare,
Public Health Service, Center for Disease Control, National Institute for Occupational Safety and
Health, Publication No. 77-206, Aug. 1977.
4. “OSHA Safety and Health Standards, General Industry,” (29 CFR 1910), Occupational Safety and
Health Administration, OSHA 2206, (Revised, January 1976).
5. “Safety in Academic Chemistry Laboratories,” American Chemical Society Publication,
Committee on Chemical Safety, 3rd Edition, 1979.
6. “Handbook for Analytical Quality Control in Water and Wastewater Laboratories,” EPA-60014-
79-019, U. S. Environmental Protection Agency, Environmental Monitoring and Support
Laboratory: Cincinnati, Ohio, March 1979.
7. ASTM Annual Book of Standards, Part 31, D3370, “Standard Practice for Sampling Water,”
American Society for Testing and Materials, Philadelphia, Pennsylvania, p. 76, 1980.
8. Burke, J.A., “Gas Chromatography for Pesticide Residue Analysis; Some Practical Aspects,”
Journal of the Association of Official Analytical Chemists, 48, 1037 (1965).
9. McNair, H.M., and Bonelli, E. J., Basic Chromatography, Consolidated Printing, Berkeley,
California, p. 52, 1969.
10. Eichelberger, J.W., Harris, L.E., and Budde, W.L. “Reference Compound to Calibrate Ion
Abundance Measurement in Gas Chromatography-Mass Spectrometry,” Analytical Chemistiy, 47,
995 (1975).
11. Glaser, J.A. et al., “Trace Analysis for Wastewaters,” Environmental Science & Technology, 15,
1426 (1981).
239
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Method 622
Table 1. Chromatographic Conditions and Estimated Method Detection Limits
GC Retention Time Estimated MDL
Paramet& Column (mu) (pg/U
Demeton la 1.16 0.25
2.53
Phorate la 1.43 0.15
Disulibton la 2.10 0.20
Trichioronate la 2.94 0.15
Fenthion la 3.12 0.10
Tokuthion la 3.40 0.5
Boislar la 4.23 0.15
Fensulfothion la 6.41 1.5
Azinphos methyl la 6.80 1.5
Coumaphos la 11.6 1.5
Dichlorvos lb 0.8 0.1
Mevhqhos lb 2.41 0.3
Stirolbs lb 8.52 5.0
Ethoprop 2 3.02 0.25
Parathion methyl 2 3.37 0.3
Ronnel 2 5.57 0.3
Chlorpyrifbs methyl 2 5.72 0.3
chlorpyrifos 2 6.16 0.3
Merphos 2 7.45 0.25
____ 2 7.73 0.6
Dichiorvos 3 1.50 0.1
NaIad 3 3.28 0.1
Stirofos 3 5.51 5.0
Column 1* Conditions: Supelcoport (100/120 mesh) coated with 5% SP-2401 packed in a 180 cm long
by 2 mm ID glass column with helium carrier gas at a flow rate of 30 mL/min. Column temperature,
programmed: Initial 150°C, hold for 1 minute, then program at 25°C/mm to 220°C and hold. A flame
photometric detector was used with this column to estinute the MDL.
Column lb Conditions: Same as Column 1*, except nitrogen carrier gas at a flow rate of 30 mL/min.
Temperature, programmed: Initial 170°C, hold 2 minutes, then program at 20°C/mm to 220°C and
hold.
Column 2 Conditions: Supelcoport (100/120 mesh) coated with 3% SP-2401 packed in a 180 cm long
by 2 mm ID glass column with helium carrier gas at a flow rate of 25 mL/min. Column temperature,
progr2mmed. initial 170°C, hold for 7 minutes, then program at 10°C/mm to 250°C and hold. A
thermionic bead detector was used with this column to estimate the MDL.
240
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Method 622
Table 1. Chromatographic Conditions and Estimated Method Detection Limits
(cont.)
Column 3 Conditions: Gas Chrom Q (100/120 mesh) coated with 15% SE-54 packed in a 50 cm long
by 1 /a-Inch OD PTFE column with nitrogen carrier gas at a flow rate of 30 mL/min. Temperature,
programmed: Initial 100°C, then program immediately at 25°C/mm to 200°C and hold. An electrolytic
detector in the halogen mode was used with this column to estimate the MDL.
Table 2. Single-Operator Accuracy and Precision
Average Standard Spike
Percent Deviation Range Number of
Parameter Recovery 1%) (pg/Li Analyses Types
Azinphos methyl 72.7 18.8 21-250 17 3
Bolstar 64.6 6.3 4.9-46 17 3
Chlorpyrifos 98.3 5.5 1.0-50.5 18 3
Coumaphos 109.0 12.7 25-225 17 3
Demeton 67.4 10.5 11.9-314 17 3
Diazinon 67.0 6.0 5.6 7 1
Dichlorvos 72.1 7.7 15.6-517 16 3
Disulfoton 81.9 9.0 5.2-92 17 3
Ethoprop 100.5 4.1 1.0-51.5 18 3
Fensulfothion 94.1 17.1 23.9-1 10 17 3
Fenthion 68.7 19.9 5.3-64 17 3
Merphos 120.7 7.9 1.0-50 18 3
Mevinphos 56.5 7.8 15.5-520 16 3
Naled 78.0 8.1 25.8-294 16 3
Parathion methyl 96.0 5.3 0.5-500 21 3
Phorate 62.7 8.9 4.9-47 17 3
Ronnel 99.2 5.6 1.0-50 18 3
Stirofos 66.1 5.9 30.3-505 16 3
Tokuthion 64.6 6.8 5.3-64 17 3
Trichloronate 105.0 18.6 20 3 1
241
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M.thod 622
C
I . .
0
H
JL
I I I I I I I I I II lIIII!IIIIIIt
0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0 11.0 12.0
Retsi lon Time (minUtes)
Figure 1. Gas Chromatogram of Organophosphorus Pesticides on Column 1 a.
For Conditions, See Table 1.
242
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Method 622
Retention Time (minutes)
Figure 2. Gas Chromatogram of Organophosphorous Pesticides on Column 1 b.
For Conditions, See Table 1.
Dichiorvos
Mevinphos
/
Stirofos
0
1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0
9.0
A -CO2-52
243
-------
M.mod 6
Chiorpyrdos
\
-——I I I I I I I I I I I I I
1.0 2.0 3.0 4.0 5.0 6.0 7.0
/ Merphos
/ Diazinon
I I I I
8.0 9.0
R nfl. —
Figure 3.
Gas Chromatogram of Organophosphorus Pesticides on Column 2.
For Conditions, See Table 1.
Bho op
\
Parathion Methyl
0
A
244
-------
Method 622
0 1.0 2.0 3.0
4.0 5.0 6.0 7.0 8.0
Figure 4. Gas Chromatogram of Organophosphorus Pesticides on
Column 3. For Conditions, See Table 1.
Stirofos
Dichiorvos
/
Na ed
/
Retention Time (minutes)
A -O -55
245
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Method 622.1
The Determination of
Thiophosphate Pesticides in
Municipal and Industrial
Waste water
-------
Method 622.1
The Determination of Thiophosphate Pesticides in Municipal and
Industrial Wastewater
SCOPE AND APPLICATION
1.1 This method covers the determination of certain thiophosphate pesticides. The following
parameters can be determined by this method:
Parameter CAS No.
Aspon 3244-90-4
Dichlofenthion 97-17-6
Famphur 52-85-7
Fenitrothion 122-14-5
Fonophos 944-22-9
Phosmet 132-11-6
Thionazin 297-97-2
1.2 This is a gas chromatographic (GC) method applicable to the determination of the compounds
listed above in municipal and industrial discharges as provided under 40 CFR 136.1. Any
modification of this method beyond those expressly permitted shall be considered a major
modification subject to application and approval of alternative test procedures under 40 CFR 136.4
and 136.5.
1.3 The method detection limit (MDL, defined in Section 15) for each parameter is listed in Table 2.
The MDL for a specific wastewater may differ from those listed, depending upon the nature of
interferences in the sample matrix.
1.4 The sample extraction and concentration steps in this method are essentially the same as in certain
other 600-series methods. Thus, a single sample may be extracted to measure the compounds
included in the scope of the methods. When cleanup is required, the concentration levels must
be high enough to permit selecting aliquots, as necessary, in order to apply appropriate cleanup
procedures.
1.5 This method is restricted to use by or under the supervision of analysts experienced in the use of
gas chromatography and in the interpretation of gas chromatograms. Each analyst must
demonstrate the ability to generate acceptable results with this method using the procedure
described in Section 8.2.
1.6 When this method is used to analyze unfamiliar samples for any or all of the compounds above,
compound identifications should be supported by at least one additional qualitative technique.
This method describes analytical conditions for a second gas chromatographic column that can be
used to confirm measurements made with the primary column. Section 14 provides gas
249
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II _________
Method 622.1
chromatograph/mass spectrometer (GC/MS) criteria appropriate for the qualitative confirmation
of compound identifications.
2. SUMMARY OF METHOD
2.1 A measured volume of sample, approximately I L, is extracted with methylene chloride using
a separatory funnel. The methylene chloride extract is dried and concentrated to 1.0 mL. Gas
chromatographic conditions are described which permit the separation and measurement of the
compounds in theextract by alkali flame detector gas chromatography (GCIAFD).’
2.2 This method provides a Florisil column cleanup procedure to aid in the elimination of
interferences that may be encountered.
3. INTERFERENCES
3.1 Method interferences may be caused by contaminants in solvents, reagents, glassware, and other
sample-processing apparatus that lead to discrete artifacts or elevated baselines in gas
chrc __. All reagents and apparatus must be routinely demonstrated to be free from
interferences under the conditions of the analysis by running laboratory reagent blanks as
described in Sectiqn 8.5.
3.1.1 Glassware must be scrupulously cleaned. 2 Clean all glassware as soon as possible after
use by thoroughly rinsing with the last solvent used in it. Follow by washing with hot
water and detergent and thoroughly rinsing with tap and reagent water. Drain dry and
heat in an oven or muffle furnace at 400°C fur 15 to 30 minutes. Do not heat volumetric
glassware. Some thermally stable materials, such as PCBs, may not be eliminated by this
treatment. Thorough rinsing with acetone and pesticide-quality hexane may be substituted
for the he ing. After drying and cooling, seal and store glassware in a clean
environment to prevent any accumulation of dust or other contaminants. Store inverted
or capped with alnmmum foil.
3.1.2 The use of high-purity reagents and solvents helps to minimize interference problems.
Purification of solvents by distillation in all-glass systems may be required.
3.2 Matrix interferences may be caused by contaminants that are coextracted from the sample. The
extent of matrix interferences will vary considerably from source to source, depending upon the
nature and diversity of the industrial complex or municipality sampled. The cleanup procedure
in Section 11 can be used to overcome many of these interferences, but unique samples may
require additional cleanup approaches to achieve the MDLs listed in Table 2.
4. SAFETY
4.1 The toxicity or carcinogenicity of each reagent used in this method has not been precisely defined;
however, each chemical compound should be treated as a potential health hazard. From this
viewpoint exposure to these chemicals must be reduced to the lowest possible level by whatever
n ans available. The laboratory is responsible for maintaining a current awareness file of OSHA
regulations regarding the safe hawfling of the chemicals specified in this method. A reference file
of material data handling sheets should also be made available to all personnel involved in the
250
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Method 622.1
chemical analysis. Additional references to laboratory safety are available and have been
identified” for the information of the analyst.
5. APPARA TUS AND MA TERIALS
5.1 Sampling equipment, for discrete or composite sampling.
5.1.1 Grab-sample bottle: Amber borosiicate or flint glass, 1-quart or 1-L volume, fitted with
screw-caps lined with PTFE. Foil may be substituted for PTFE if the sample is not
corrosive. If amber bottles are not available, protect samples from light. The container
and cap liner must be washed, rinsed with acetone or methylene chloride, and dried
before use to minimize cont2mination.
5.1.2 Automatic sampler (optional): Must incorporate glass sample containers for the
collection of a minimum of 250 mL. Sample containers must be kept refrigerated at 4°C
and protected from light during compositing. If the sampler uses a peristaltic pump, a
minimum length of compressible silicone rubber tubing may be used. Before use,
however, the compressible tubing should be thoroughly rinsed with methanol, followed
by repeated rinsing with distilled water to minimize the potential for contamination of the
sample. An integrating flow meter is required to collect flow-proportional composites.
5.2 Glassware. (All specifications are suggested. Catalog numbers are included for illustration only.)
5.2.1 Separatory funnel: 2000-mL, with PTFE stopcock.
5.2.2 Drying column: Chromatographic column 400 mm long by 10 mm ID with coarse fit.
5.2.3 Chromatographic column: 400 mm long by 19 mm ID with 250-mL reservoir at the top
and PTFE stopcock (Kontes K-420290 or equivalent).
5.2.4 Concentrator tube, Kuderna-Danish: 10-mL, graduated (Kontes K-570050-1025 or
equivalent). Calibration must be checked at the volumes employed in the test. A
ground-glass stopper is used to prevent evaporation of extracts.
5.2.5 Evaporative flask, Kuderna-Danish: 500-niL (Kontes K-570001-0500 or equivalent).
Attach to concentrator tube with springs.
5.2.6 Snyder column, Kuderna-Danish: Three-ball macro (Kontes K-503000-0121 or
equivalent).
5.2.7 Snyder column, Kuderna-Danbth: Two-ball micro (Kontes K-569001-0219 or
equivalent).
5.2.8 Vials: Amber glass, 10- to 15-mL capacity with PTFE-lined screw-cap.
5.2.9 Erlenmeyer flask: 250-mL.
5.2.10 Graduated cylinder: 1000-mL.
5.3 Boiling chips: Approximately 10/40 mesh carborundum. Heat at 400°C for 4 hours or extract
in a Soxhiet with methylene chloride.
5.4 Water bath: Heated, capable of temperature control (±2°C). The bath should be used in a hood.
5.5 Balance: Analytical, capable of accurately weighing to the nearest 0.0001 g.
251
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Method 622.1
5.6 Gas chromatograph: Analytical system complete with gas chromatograph suitable for on-column
injection and all required accessories including syringes, analytical columns, gases, detector, and
strip-chart recorder. A data system is recommended for measuring peak areas.
5.6.1 Column 1: 180 cm long by 2 mm ID glass, packed with 3% SP-2250 on Supelcoport
(100/120 mesh) or equivalent. This column was used to develop the method performance
statements in Section 15. Alternative columns may be used in accordance with the
provisions described in Section 12.1.
5.6.2 Column 2: 180 cm long by 2 mm ID glass, packed with 3% SP-2100 on Supelcoport
(100/120 mesh) or equivalent.
5.6.3 Detector: Alkali flame detector (AFD), sometimes referred to as a nitrogen-phophorous
detector (NPD) or a thermionic-specific detector (TSD). This detector has proven
effective in the analysis of wastewaters for the compounds listed in the scope and was
used to develop the method performance statements in Section 15.
6. REAGENTS
6.1 Reagent water: Reagent water is defined as a water in which an interferent is not observed at the
method detection $imit of each parameter of interest.
6.2 Methylene chloride, methanol, petroleum ether, anhydrous ethyl ether, and acetone:
Distilled-in-glass quality or equivalent. Ethyl ether must be free of peroxides as indicated by EM
Quant Test Strips (available from Scientific Products Co., Catalog No. P 1126-8 and other
suppliers). Procedures recommended for removal of peroxides are provided with the test strips.
6.3 Sodium sulfate: ACS, granular, anhydrous; heated in a muffle furnace at 400°C overnight.
6.4 Florisil: PR grade (60/100 mesh). Purchase activated at 675°C and store in a brown glass bottle.
To prepare for use, place 150 g in a wide-mouth jar and heat overnight at 160 to 170°C. Seal
lightly with FIFE w aluminum-foil-lined screw-cap and cool to room temperature.
6.5 6N sodium hydroxide.
6.6 6N sulfuric acid.
6.7 Stock standard solutions (1.00 g ig/ iL): Stock standard solutions can be prepared from pure
standard materials or purchased as certified solutions.
6.7.1 Prepare stock standard solutions by accurately weighing about 0.0100 g of pure material.
Dissolve the material in distilled-in-glass quality ethyl ether and dilute to volume in a
10-mL volumetric flask. Larger volumes can be used at the convenience of the analyst.
If compound purity is certified at 96% or greater, the weight can be used without
correction to calculate the concentration of the stock standard. Commercially prepared
stock standards can be used at any concentration if they are certified by the manufacturer
or by an independent source.
6.7.2 Transfer the stock standard solutions into PTFE-sealed screw-cap bottles. Store at 4°C
and protect from light Frequently check standard solutions for signs of degradation or
evaporation, especially just prior to preparing calibration standards from them.
6.7.3 Stock standard solutions must be replaced after 6 months, or sooner if comparison with
check standards indicates a problem.
252
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Method 622.1
7. CALIBRATION
7.1 Establish gas chromatographic operating parameters equivalent to those indicated in Table 2. The
gas chromatographic system can be calibrated using the external standard technique (Section 7.2)
or the internal standard technique (Section 7.3).
7.2 External standard calibration procedure:
7.2.1 For each compound of interest, prepare calibration standards at a minimum of three
concentration levels by adding volumes of one or more stock standards to a volumetric
flask and diluting to volume with ethyl ether. One of the external standards should be
at a concentration near, but above, the method detection limit. The other concentrations
should correspond to the range of concentrations expected in the sample concentrates or
should define the working range of the detector.
7.2.2 Using injections of 1 to 5 L of each calibration standard, tabulate peak height or area
responses against the mass injected. The results can be used to prepare a calibration
curve for each compound. Alternatively, the ratio of the response to the mass injected,
defined as the calibration factor (CF), can be calculated for each compound at each
standard concentration. If the relative standard deviation of the calibration factor is less
than 10% over the working range, the average calibration factor can be used in place of
a calibration curve.
7.2.3 The working calibration curve or calibration factor must be verified on each working
shift by the measurement of one or more calibration standards. If the response for any
compound varies from the predicted response by more than ± 10%, the test must be
repeated using a fresh calibration standard. Alternatively, a new calibration curve or
calibration factor must be prepared for that compound.
7.3 Internal standard calibration procedure: To use this approach, the analyst must select one or more
internal standards similar in analytical behavior to the compounds of interest. The analyst must
further demonstrate that the measurement of the internal standard is not affected by method or
matrix interferences. Due to these limitations, no internal standard applicable to all samples can
be suggested.
7.3.1 Prepare calibration standards at a minimum of three concentration levels for each
compound of interest by adding volumes of one or more stock standards to a volumetric
flask. To each calibration standard, add a known constant amount of one or more
internal standards, and dilute to volume with ethyl ether. One of the standards should
be at a concentration near, but above, the method detection limit. The other
concentrations should correspond to the range of concentrations expected in the sample
concentrates or should define the working range of the detector.
253
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Method 622.1
7.3.2 Using injections of 1 to 5 L of each calibration standard, tabulate the peak height or
area responses against the concentration for each compound and internal standard.
Calculate response factors (RF) for each compound as follows:
Equation 1
= ( A,)(C , )
(A 11 )(C,)
where
A, = Response for the parameter to be measured
A 1 = Response for the internal standard
C 1 = Concentration of the Internal stwidanl, In pgIL
C, = Concentration of the parameter to be measured, In &gIL
If the RF yalue over the working range is constant, less than 10% relative standard
deviation, the RF can be assumed to be invariant and the average RF can be used for
calculations. Alternatively, the results can be used to plot a calibration curve of response
ratios, A,/A 1 against RF.
7.3.3 The working calibration curve or RF must be verified on each working shift by the
measurement of one or more calibration standards. If the response for any compound
varies from the predicted response by more than ± 10%, the test must be repeated using
a fresh calibration standard. Alternatively, a new calibration curve must be prepared for
that compound.
7.4 Before using any cleanup procedure, the analyst must process a series of calibration standards
through the procedure to validate elution patterns and the absence of interferences from the
reagents.
8. QUAU7Y COivmoL
81 Pith laboratory n*ang this method is.required to operate a formal quality control program. The
flhiflh dm requiruneEds of this program consist of an initial demonstration of laboratory capability
and the analysis of spiked samples as a continuing check on performance. The laboratory is
required to mah ain performance records to define the quality of data that is generated.
81.1 BefOre performing any analyses, the analyst must demonstrate the ability to generate
acceptable accuracy and precision with this method. This ability is established as
described in Section 8.2.
8.1.2 In recognition of the rapid advances occurring in chromatography, the analyst is
permitted certain options to improve the separations or lower the cost of measurements.
Each time such modifications to the m hod are made, the analyst is required to repeat
the procedure in Section 8.2.
254
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Method 622.1
8.1.3 The laboratory must spike and analyze a minimum of 10% of all samples to monitor
continuing laboratory performance. This procedure is described in Section 8.4.
8.2 To establish the ability to generate acceptable accuracy and precision, the analyst must perform
the following operations.
8.2.1 Select a representative spike concentration for each compound to be measured. Using
stock standards, prepare a quality control check sample concentrate in methanol, 1000
times more concentrated than the selected concentrations.
8.2.2 Using a pipette, add 1.00 mL of the check sample concentrate to each of a minimum of
four 1000-mL aliquots of reagent water. A representative wastewater may be used in
place of the reagent water, but one or more additional aliquots must be analyzed to
determine background levels, and the spike level must exceed twice the background level
for the test to be valid. Analyze the aliquots according to the method beginning in
Section 10.
8.2.3 Calculate the average percent recovery (R) and the standard deviation of the percent
recovery (s) for the results. Wastewater background corrections must be made before
R and s calculations are performed.
8.2.4 Using the appropriate data from Table 3, determine the recovery and single-operator
precision expected for the method, and compare these results to the values measured in
Section 8.2.3. If the data are not comparable, the analyst must review potential problem
areas and repeat the test.
8.3 The analyst must calculate method performance criteria and define the performance of the
laboratory for each spike concentration and parameter being measured.
8.3.1 Calculate upper and lower control limits for method performance as follows:
Upper Control Limit (UCL) = R + 3s
Lower Control Limit (LCL) = R -3s
where R and s are calculated as in Section 8.2.3. The UCL and LCL can be used to
construct control charts that are useful in observing trends in performance.
8.3.2 The laboratory must develop and maintain separate accuracy statements of laboratory
performance for wastewater samples. An accuracy statement for the method is defined
as R ± s. The accuracy statement should be developed by the analysis of four aliquots
of wastewater as described in Section 8.2.2, followed by the calculation of R and s.
Alternately, the analyst may use four wastewater data points gathered through the
requirement for continuing quality control in Section 8.4. The accuracy statements should
be updated regularly. 6
8.4 The laboratory is required to collect in duplicate a portion of their samples to monitor spike
recoveries. The frequency of spiked sample analysis must be at least 10% of all samples or one
sample per month, whichever is greater. One aliquot of the sample must be spiked and analyzed
as described in Section 8.2. If the recovery for a particular compound does not fall within the
control limits for method performance, the results reported for that compound in all samples
255
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Method 622.1
processed as part of the same set must be qualified as described in Section 13.3. The laboratory
should monitor the frequency of data so qualified to ensure that it remains at or below 5%.’
8.5 Before processing any samples, the analyst should demonstrate though the analysis of a 1-L aliquot
of reagent water that all glassware and reagent interferences are under control. Each time a set
of samples is extracted or there is a change in reagents, a laboratory reagent blank should be
processed as a safequard against laboratory contamination.
8.6 It is recommended that the laboratory adopt additional quality assurance practices for use with this
method. The specific practices that are most productive depend upon the needs of the laboratory
and the nature of the samples. Field duplicates may be analyzed to monitor the precision of the
sampling technique. When doubt exists over the identification of a peak on the chromatogram,
confirmatory techniques such as gas chromatography with a dissimilar column, specific element
detector, or mass spectrometer must be used. Whenever possible, the laboratory should perform
analysis of standard reference materials and participate in relevant performance evaluation studies.
9. SAMPLE COLLECTiON, PRESERVATiON. AND HANDLING
9.1 Grab samples must be collected in glass containers. Conventional sampling practices 7 should be
followed; however, the bottle must not be prerinsed with sample before collection. Composite
samples should be collected in refrigerated glass containers in accordance with the requirements
of the program. Automatic sampling equipment must be as free as possible of plastic and other
potential sources of contamination.
9.2 The samples must be iced or refrigerated at 4°C from the time of collection until extraction.
9.3 Adjust the pH of the sample to 6 to 8 with 6N sodium hydroxide or 6N sulfuric acid immediately
after sampling.
10. SAMPLE EXTRACTiON
10.1 Mark the water meniscus on the side of the sample bottle for later determination of sample
volume. Pour the entire sample into a 2-L separatory funnel. Check the pH of the sample with
wide range pH paper and adjust to 6 to 8 with 6N sodium hydroxide or 6N sulfuric acid.
10.2 Add 60 mL of methylene chloride to the sample bottle, seal, and shake 30 seconds to rinse the
lamer walls. Transfer the solvent to the separatory funnel and extract the sample by shaking the
funnel for 2 minutes with periodic venting to release excess pressure. Allow the organic layer to
separate from the water phase for a minimum of 10 minutes. If the emulsion interface betwen
layers is more than one-third the volume of the solvent layer, the analyst must employ mechanical
techniques to complete the phase separation. The optimum technique depends upon the sample,
but may include stirring, filtration of the emulsion through glass wool, centrifugation, or other
physical methods. Collect the methylene chloride extract in a 250-mL Erlenmeyer flask.
10.3 Add a second 60-mL volume of methylene chloride to the sample bottle and repeat the extraction
procedure a second time, combining the extracts in the Erlenmeyer flask. Perform a third
extraction in the same manner.
10.4 Aasemble a Kuderna-Danish (K-D) concentrator by attaching a I0-mL concentrator tube to a
l0-mL evaporative flask. Other concentration devices or techniques may be used in place of the
K-I) if the requirements of Section 8.2 are met.
256
-------
Method 622.1
10.5 Pour the combined extract through a drying column containing about 10 cm of anhydrous sodium
sulfate, and collect the extract in the K-I) concentrator. Rinse the Erlenmeyer flask and column
with 20 to 30 mL of methylene chloride to complete the quantitative transfer. Once the flask rinse
has passed through the drying column, rinse the column with 30 to 40 mL of methylene chloride.
10.6 Add one or two clean boiling chips to the evaporative flask and attach a three-ball Snyder column.
Prewet the Snyder column by adding about I mL methylene chloride to the top. Place the K-D
apparatus on a hot water bath, 60 to 65°C, so that the concentrator tube is partially immersed in
the hot water and the entire lower rounded surface of the flask is bathed with hot vapor. Adjust
the vertical position of the apparatus and the water temperature as required to complete the
concentration in 15 to 20 minutes. At the proper rate of distillation, the bails of the column will
actively chatter, but the chambers will not flood with condensed solvent. When the apparent
volume of liquid reaches 1 mL, remove the K-D apparatus and allow it to drain and cool for at
least 10 minutes.
10.7 Remove the macro-Snyder column and rinse the flask and its lower joint into the concentrator tube
with 1 to 2 mL of methylene chloride. Add one or two clean boiling chips and attach a two-ball
micro-Snyder column to the concentrator tube. Prewet the micro-Snyder column with methylene
chloride and concentrate the solvent extract as before. When an apparent volume of 0.5 mL is
reached, or the solution stops boiling, remove the K-D apparatus and allow it to drain and cool
for 10 minutes.
10.8 Remove the micro-Snyder column and adjust the volume of the extract to 1.0 mL with methylene
chloride. Stopper the concentrator tube and store refrigerated if further processing will not be
performed immediately. If the extract is to be stored longer than 3 days, transfer the extract to
a screw-capped vial with a PTFE-lined cap. If the sample extract requires no further cleanup,
proceed with the gas chromatographic analysis in Section 12. if the sample requires cleanup,
proceed to Section 11.
10.9 Determine the original sample volume by refilling the sanple bottle to the mark and transferring
the water to a 1000-mL graduated cylinder. Record the sample volume to the nearest 5 mL.
11. CLEANUP AND SEPARA T1ON
11.1 Cleanup procedures may not be necessary for a relatively clean sample matrix. The cleanup
procedure recommended in this method has been used for the analysis of various clean waters and
industrial effluents. If particular circumstances demand the use of an alternative cleanup
procedure, the analyst must determine the elution profile and demonstrate that the recovery of
each compound of interest is no less than that reported in Table 3.
11.2 The following Florisil column cleanup procedure has been demonstrated to be applicable to the
seven thiophosphate pesticides listed in Table 1.
11.2.1 Add 20 g of Florisil to 100 mL of ethyl ether and 400 pL of reagent water in a 250-mL
Erlenmeyer flask. Shake vigorously for 15 minutes. Transfer the slurry to a
chromatographic column (Florisil may be retained with a plug of glass wool). Allow the
solvent to elute from the column until the Florisil is almost exposed to the air. Wash the
column with 25 mL of petroleum ether. Use a column flow of 2 to 2.5 mL/min
throughout the wash and elution profiles. Add an additional 50 mL of petroleum ether
to the head of the column.
257
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Method 622.1
11.2.2 Quantitatively add the sample extract from Section 10.8 to the head of the column.
Allow the solvent to elute from the column until the Florisil is almost exposed to the air.
Elute the column with 50 mL of 6% ethyl ether in petroleum ether. Discard this
fraction.
11.2.3 Elute the column with 50 mL of 15% ethyl ether n petroleum ether (Fraction 1) and
collect eluate in a K-D apparatus. Repeat process with 50 mL of 50% ethyl ether in
petroleum ether (Fraction 2), 50 mL of 100% ethyl ether (Fraction 3), 50 mL 6%
acetone in ethyl ether (Fraction 4), and 100 mL 15% acetone in ethyl ether (Fraction 5),
collecting each fraction in a separate K-D apparatus. The elution patterns for the
thiophosphates are shown in Table 1. Concentrate each fraction to 1 mL as described
in Sections 10.6 and 10.7. Proceed with gas chromatographic analysis.
11.2.4 The ab mentioned fractions can be combined before concentration at the discretion of
the analyst.
12. GAS CImOMA TOGRAPHY
12.1 Table 2 summarizes the recommended operating conditions fbi the gas chromatograph. Included
in this table are e im ted retention times and method detection limits that can be achieved by this
method. Examples of the separations achieved by Column 1 and Column 2 are shown in
Figures 1 and 2. Other packed columns, chromatographic conditions, or detectors may be used
if the requirements of Section 8.2 are met. Capillary (open-tubular) columns may also be used
if the relative standard deviations of responses for replicate injections are demonstrated to be less
than 6% and the requirements of Section 8.2 are met.
12.2 Calibrate the gas chroinatographic system daily as described in Section 7.
12.3 If the internal standard approach is being used, the analyst must not add the internal standard to
the s n le extracts until immediately before injection into the instrument. Mix thoroughly.
12.4 Inject I to 5 g&L of the sample extract using the solvent flush technique. Record the volume
injected to the nearest 0.05 pL, and record the resulting peak sizes in area or peak height units.
An automated system that consistently injects a constant volume of extract may also be used.
12.5 The width of the retention-time window used to make identifications should be based upon
measurements of actual retention-time variations of standards over the course of a day. Three
times the standard deviation of a retention-time for a compound can be used to calculate a
suggested window size; however, the experience ofihe analyst should weigh heavily in the
LMqwet.tion of chromatograms.
12.6 If the respoese for the peak exceeds the working range of the system, dilute the extract and
rean yze.
12.7 If the measurement of the peak response is prevented by the presence of interferences, fuilher
is required.
258
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Method 622.1
13. CALCULA T1ONS
13.1 Determine the concentration of individual compounds in the sample.
13.1.1 If the external standard calibration procedure is used, calculate the amount of material
injected from the peak response using the calibration curve or calibration factor in
Section 7.2.2. The concentration in the sample can be calculated as follows:
Equation 2
Concentration, igIL = ______
where
A = Amount of material injected, in ng
= Volume of extract injected, in pL
V 1 = Voiwne of total extract, in L
V , = Volume of water extracted, in mL
13.1.2 If the internal standard calibration procedure was used, calculate the concentration in the
sample using the response factor (RF) determined in Section 7.3.2 as follows:
Equation 3
(A)(I)
Concentration, gIL = (A) ,g(V)
where
A, = Response for parameter to be neasured
A 1 , = Response for the internal standard
I , = Amount of internal standard added to each extract,
in
&g
V 0 = Volume of water extracted, in L
13.2 Report results in micrograms per liter without correction for recovery data. When duplicate and
spiked samples are analyzed, report all data obtained with the sample results.
13.3 For samples processed as part of a set where the laboratory spiked sample recovery falls outside
of the control limits in Section 8.3, data for the affected compounds must be labeled as suspect.
14. GC/MS CONFIRMA T1ON
14.1 It is recommended that GC/MS techniques be judiciously employed to support qualitative
identifications made with this method. The mass spectrometer should be capable of scanning the
mass range from 35 amu to a mass 50 amu above the molecular weight of the compound. The
259
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Method 622.1
instrument must be capable of scanning the mass range at a rate to produce at least S scans per
peak but not to exceed 7 seconds per scan utilizing a 70 V (nominal) electron energy in the
electron impact ionization mode. A GC to MS interface constructed of all glass or glass-lined
materials is recommended. When using a fused-silica capillary column, the column outlet should
be threaded through the interface to within a few milimeters of the entrance to the source
ionization chamber. A computer system should be interfaced to the mass spectrometer that allows
the continuous acquisition storage on machine-readable media of all mass spectra obtained
throughout the duration of the chromatographic program.
14.2 Gas chromatographic columns and conditions should be selected for optimum separation and
performance. The conditions selected must be compatible with standard GCIMS operating
practices. Chromatographic tailing factors of less than 5.0 must be achieved.’ 0
14.3 At the beginning of each day that confirmatory analyses are to be performed, the GC/MS system
must be checked to see that all DFTPP performance criteria are achieved. 9
14.4 To confirm an identification of a compound, the background-corrected mass spectrum of the
compound must be obtained from the sample extract and compared with a mass spectrum from
a stock or calibration standard analyzed under the same chromatographic conditions. It is
recommended that at least 25 ng of material be injected into the GC/MS. The criteria below must
be met for qualitative confirmation.
14.4.1 The molecular ion and all other ions that are present above 10% relative abundance in
the mass spectrum of the standard must be present in the mass spectrum of the sample
with agreement to ± 10%. For example, if the relative abundance of an ion is 30% in
the mass spectrum of the standard, the allowable limits for the relative abundance of that
ion inthemass spectrum forthesamplewould be2Oto 40%.
14.4.2 The retention time of the compound in the sample must be within 6 seconds of the same
compound in the standard solution.
14.4.3 Compounds that have very similar mass spectra can be explicitly identified by GC/MS
only on the basis of retention time data.
14.5 Where available, chemical ionization mass spectra may be employed to aid in the qualitative
identification process.
14.6 Should these MS procedures fail to provide satisfactory results, additional steps may be taken
before reanalysis. These may include the use of alternative packed or capillary GC columns or
additional cleanup (Section 11).
15. MEmoo PERFORMANCE
15.1 The method detection limit (MDL) is defined as the minimum concentration of a substance that
can be measured and reported with 99% confidence that the value is above zero. ’ The MDL
concentrations listed in Table 2 were obtained using reagent water.’ Similar results were achieved
using representative wastewaters.
15.2 This method has been tested for linearity of recovery from spiked reagent water and has been
demonstrated to be applicable over the concentration range from 10 x MDL to 1000 x MDL.
15.3 In a single laboratory, Battelle Columbus Laboratories, using spiked wastewater samples, the
average recoveries presented in Table 3 were obtained alter Floristi cleanup Seven replicates of
260
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Method 622.1
each of two different wastewaters were spiked and analyzed. The standard deviation of the
percent recovery is also included in Table 3•1
261
-------
Method 622.1
References
1. i)evelopment of Methods for Pesticides in Wastewaters, EPA Contract Report 68-03-2956 (In
preparation).
2. ASTM Annual Book of Standards, Part 31, D3694, TM Standard Practice for Preparation of Sample
Containers and for Preservation,N American Society for Testing and Materials, Philadelphia,
Pennsylvania, p. 679, 1980.
3. Carcinogens - Working with Carcinogens, Department of Health, Education, and Welfare,
Public Health Service, Center for Disease Control, National Institute for Occupational Safety and
Health, Publication No. 77-206, August, 1977.
4• OSHA Safety and Health Standards, General Industry,N (29 CFR 1910), Occupational Safety and
Health Administration, OSHA 2206 (Revised, January 1976).
5. Safety in Academic Chemistry Laboratories, American Chemical Society Publication,
Committee on Chemical Safety, 3rd Edition, 1979.
6. ‘Handbook for Analytical Quality Control in Water and Wastewater Laboratories,
EPA-600/4-79-019, U. S. Environmental Protection Agency, Environmental Monitoring and
Support Laboratory - Cincinn*i, Cbio, March 1979.
7. ASTM Annual Book of Standards, Part 31, D3370, ‘Standard Practice for Sampling Water,’
American Society for Testing and Materials,Philadelphia, Pennsylvania, p. 76, 1980.
8. Burke, J.A., ‘Gas Chromatography for Pesticide Residue Analysis; Some Practical Aspects,’
Journal q ’the Associadon Official Analytical Ojemists, 48, 1037 (1965).
9. Eichelberger, J.W., Harris, L. E., and Budde, W.L., ‘Reference Compound to Calibrate Ion
Abundance Measurement in Gas Chromatography Mass Spectrometry,’ Analytical (Yzemlstiy, 47,
995 (1975).
10. McNalr, H.M., and Bonelli, EJ., Basic Ovonsatography, Consolidated Printing, Berkeley,
California, p. 52 (1969).
11. Glaser, l.A. et al, ‘Trace Analysis for Wastewaters’, Envlro,unental Science and Technology, 15,
1426(1981).
262
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Method 622.1
Table 1. Elution Orders and Recoveries of Thiophosphates from Florisil
Recovery in Specified Fraction. % (a)
Compound Fl F2 F3 F4 F5 F6 F7 Total
Aspon 94 2 96
Dichlofenthion 92 92
Famphur 6 103 109
Fenitrothion 51 55 106
Fonophos 84 6’ 90
Phosmet 37 69 106
Thionazine 2 93 95
(a) Results of single determination with 100 &g of each compound. Elution solvents were 50 mL each
of the following:
Fl = 2% methylene chloride in petroleum ether
F2 =6% ethyl ether in petroleum ether
F3 = 15% ethyl ether in petroleum ether
F4 = 50% ethyl ether in petroleum ether
F5 = 100% ethyl ether
F6 = 6% acetone in ethyl ether
Fl = 15% acetone in ethyl ether
263
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Method 622.1
Table 2. Chromatographic Conditions and Estdimated Method Detection Limits
Retention Time (Minutes)
Parametet Column 1 Column 2 MDL
(pg/L)
Thionazin 18.3 25.0 1
Fonophos 20.5 27,8 0.7
Dichlofrnthion 21.4 29.4 0.7
Aspon 22.6 30.2 0.6
Fenitrothion 23.1 30.8 2
Famphur 28.1 34.8 19
Phosmet 30.0 36.2 1
Column 1 conditions: Supelcoport (100/120 mesh) coated with 3% SP-2250 packed in a 1.8 m long by
2 mm ID glass column with helium carrier gas at a flow rate of 30 mL/min. Column temperature is
programmed from 80°C to 300°C at 8°C/mm with a 4 minute hold at each extreme, injector temperature
is 250°C and detector is 300°C. Alkali flame detector at bead voltage of 16 volts.
Column 2 conditions: Supelcoport (100/120 mesh) coated with 3% SP-2100 packed in a 1.8 m long by
2 mm ID glass columns with helium carrier gas at a flow rate of 30 mL/minute. Column temperature
is programmed from 80°C to 300°C at 8°C/mm with a 4 minute hold at each extreme, injector
temperature is 250°C and detector is 300°C.
264
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Method 622.1
Table 3. Single-Laboratory Accuracy and Precision (a)
Mean % Relative
Sample Background Spike Recovery Standard Number of
Parameter Type (b) (pg/L) (c i (pg/Li % Deviation Replicates
Aspon 1 ND 50 83 7 7
2 ND 500 87 3 7
Dichlofenthion 1 ND 50 83 7 7
2 ND 500 84 4 7
Famphur 1 ND 50 86 6 7
2 ND 500 86 4 7
Fenitrothion I ND 50 82 7 7
2 ND 500 83 4 7
Fonophos 1 ND 50 84 7 7
2 ND 500 86 4 7
Phosmet 1 ND 50 85 5 7
2 ND 500 87 5 7
Thionizin 1 ND 50 84 7 7
2 ND 500 89 5 7
(a) Column 1 conditions were used.
(b) I = Low-level relevant industrial effluent
2 = Municipal sewage influent
(c) ND = Not detected
265
-------
? kthod 62 1
Fonolos
/
Thbna n D b
\ / MPon
v 17
Il 1’
II Phosmet
—/ / I sLT I I I I I I
0 17.0 19.0 21.0 23.0 25.0 27.0 29.0 31.0 33.0 35.0
Retention Time (minutes)
A52 OO2 6
Figwe 1. GC-AFD Chromatogram of 100 ng Each of Seven Thiophosphates (Column 1).
266
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Method 622.1
A52-002-57
Figure 2. GC-FID Chromatogram of 100 ng Each of Seven Thiophosphates (Column 2).
Fonofos
/
Dichiotenthion
Aspon
Famphur
/
Fenhtrothion
/r
0 25.5
I I I I
27.0 28.5
I I I I -— I
30.0 31.5 33.0
Retention Time (minutes)
I I
34.5
I I I I I I
36.0 37.5 39.0
267
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Method 627
The Determination of
Dinitroaniline Pesticides in
Municipal and Industrial
Waste water
-------
Method 627
The Determination of Dinitroani/ine Pesticides in Municipal and
Industrial Wastewater
SCOPE AND APPLICATiON
1.1 This method covers the determination of certain dinitroaniline pesticides. The following
parameters can be determined by this method:
Parameter STORET No. CAS No.
Benfiuralin 39002 1861-40-1
Ethalfluralin 55283-68-6
Isopropalin 33820-53-0
Profiuralin 26399-36-0
Trifluralin 39030 1582-09-8
1.2 This method fails to distinguish between benfluralin, ethalfiuralin, and trifluralin. When more
than one of these materials may be present in a sample, the results are reported as trifiuralin.
1.3 This is a gas chromatographic (CiC) method applicable to the determination of the compounds
listed above in industrial and municipal discharges as provided under 40 CFR 136.1. Any
modification of this method beyond those expressly permitted shall be considered a major
modification subject to application and approval of alternative test procedures under 40 CFR 136.4
and 136.5.
1.4 The method detection limits (MDL, defined in Section 15) for four of the parameters are listed
in Table 1. The MDL for a specific wastewater may differ from those listed, depending upon the
nature of interferences in the sample matrix.
1.5 The sample extraction and concentration steps in this method are essentially the same as several
other the 600-series methods. Thus, a single sample may be extracted to measure the parameters
included in the scope of each of these methods. When cleanup is required, the concentration
levels must be high enough to permit selecting aliquots, as necessary, in order to apply appropriate
cleanup procedures. Under gas chromatography, the analyst is allowed the latitude to select
chromatographic conditions appropriate for the simultaneous measurement of combinations of
these parameters (see Section 12).
1.6 This method is restricted to use by or under the supervision of analysts experienced in the use of
gas chromatography and in the interpretation of gas chromatograms. Each analyst must
demonstrate the ability to generate acceptable results with this method using the procedure
described in Section 8.2.
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MetlwxI 627
1.7 When this method is used to analyze unfamiliar samples for any or all of the compounds above,
compound identifications should be supported by at least one additional qualitative technique.
This method describes analytical conditions for a second gas chromatographic column that can be
used to confirm measurements made with the primary column within the limitations described in
Section 1.2. Section 14 provides gas chromatograph/mass spectrometer (GC/MS) criteria
appropriate for the qualitative confirmation of compound identifications.
2. SuMi’wv OF METHOD
2.1 A measured volume of sample, approximately 1 L, is extracted with 15% methylene chloride
using a separatory funnel. The methylene chloride extract is dried and exchanged to hexane
during concentration to a volume of 10 mL or less. Gas chromatographic conditions are described
which permit the separation and measurement of the compounds in the extract by electron capture
(EC) gas chromatography. 1
2.2 This method provides an optional Florisil column cleanup procedure to aid in the elimination or
reduction of interferences which may be encountered.
3. INTERFERENCES
3.1 Method interferences may be caused by contaminants in solvents, reagents, glassware, and other
sample-processing apparatus that lead to discrete artifacts or elevated baselines in gas
chromatograms. All reagents and apparatus must be routinely demonstrated to be free from
interferences under the conditions of the analysis by running laboratory reagent blanks as
described in Section 8.5.
3.1.1 Glassware must be scrupulously cleaned. 2 Clean all glassware as soon as possible after
use by thoroughly rinsing with the last solvent used in it. Follow by washing with hot
water and detergent and thorough rinsing with tap and reagent water. Drain dry, and heat
in an oven or muffle furnace at 400°C for 15 to 30 minutes. Do not heat volumetric
ware. Thermaily stable materials, such as PCBs, may not be eliminated by this
treatment. Thorough rinsing with acetone and pesticide-quality hexane may be substituted
for the heating. After drying and cooling, seal and store glassware in a clean
environment to prevent any accumulation of dust or other contaminants. Store inverted
or capped with aluminum foil.
3.1.2 The use of high-purity reagents and solvents helps to minimize interference problems.
Purification of solvents by distillation in all-glass systems may be required.
3.2 Interferences by phthalate esters can pose a major problem in pesticide analysis when the EC
detector is used. These compounds generally appear in the chromatogram as large, late-eluting
peaks. Common flexible plastics contain varying amounts of phthalates. These phthalates are
easily extracted or leached from such materials during laboratory operations. Cross-contamination
of clean glassware occurs when plastics are handled during extraction steps, especially when
solvent-wetted surfaces are handled. Interferences from phthalates can be minimized by avoiding
the use of plastics in the laboratory. Exhaustive cleanup of reagents and glassware may be
required to elbnin*e background phthalate contamination. 34
272
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Method 627
3.3 Matrix interferences may be caused by contaminants that are coextracted from the sample. The
extent of matrix interferences will vary considerably from source to source, depending upon the
nature and diversity of the industrial complex or municipality sampled. Unique samples may
require special cleanup approaches to achieve the MDL listed in Table 1.
4. SAFETY
4.1 The toxicity or carcinogenicity of each reagent used in this method has not been precisely defined;
however, each chemical compound must be treated as a potential health hazard. From this
viewpoint, exposure to these chemicals must be reduced to the lowest possible level by whatever
means available. The laboratory is responsible for maintaining a current awareness file of OSHA
regulations regarding the safe handling of the chemicals specified in this method. A reference file
of material data handling sheets should also be made available to all personnel involved in the
chemical analysis. Additional references to laboratory safety are available and have been
identified 7 for the information of the analyst.
5. APPARATUS AND MA TERIALS
5.1 Sampling equipment, for discrete or composite sampling.
5.1 .1 Grab-sample bottle: Amber borosilicate or flint glass, 1-L or 1-quart volume, fitted with
screw-caps lined with TFE-fluorocarbon. Aluminum foil may be substituted for TFE if
the sample is not corrosive. If amber bottles are not available, protect samples from
light. The container and cap liner must be washed, rinsed with acetone or methylene
chloride, and dried before use to minimize contamination.
5.1.2 Automatic sampler (optional): Must incorporate glass sample containers for the
collection of a minimum of 250 mL. Sample containers must be kept refrigerated at 4°C
and protected from light during compositing. If the sampler uses a peristaltic pump, a
minimum length of compressible silicone rubber tubing may be used. Before use,
however, the compressible tubing must be thoroughly rinsed with methanol, followed by
repeated rinsings with reagent water to minimize the potential for contamination of the
sample. An integrating flow meter is required to collect flow-proportional composites.
5.2 Glassware. (All specifications are suggested. Catalog numbers are included for illustration only.)
5.2.1 Separatory funnel: 2000-mL, with TFE-fluorocarbon stopcock, ground-glass or TFE
stopper.
5.2.2 Drying column: Chromatographic column 400 mm long by 19 mm II) with coarse-fitted
disc.
5.2.3 Concentrator tube, Kuderna-Danish: 10-mL, graduated (Kontes K-570050-1025 or
equivalent). Calibration must be checked at the volumes employed in the test. Ground-
glass stopper is used to prevent evaporation of extracts.
5.2.4 Evaporative flask, Kuderna-Danish: 500-mi (Kontes K-570001 -0500 or equivalent).
Attach to concentrator tube with springs.
5.2.5 Snyder column, Kuderna-Danish: Three-ball macro (Kontes K-503000-0121 or
equivalent).
273
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Method 627
5.2.6 Vials: Amber glass, 10. to 15-mL capacity with TFE-fluorocarbon-lined screw-cap.
5.3 Boiling chips: approximately 10/40 mesh. Heat at 400°C for 30 minutes or perform a Soxhiet
extraction with methylene chloride.
5.4 Water bath: Heated, with concentric ring cover, capable of temperature control (±2°C). The
bath should be used in a hood.
5.5 Balance: Analytical, capable of accurately weighing to the nearest 0.0001 g.
5.6 Gas chromatograph: Analytical system complete with gas chromatograph suitable for on-column
injection and all required accessories including syringes, analytical columns, gases, detector, and
strip-chart recorder. A data system is recommended for measuring peak areas.
5.6.1 Column 1: 180 cm long by 2 mm ID glass, packed with 1.5% OV-1711.95% OV-210
on Gas Cbrom Q (100/120 mesh) or equivalent. This column was used to develop the
method performance statements in Section 15. Alternative columns may be used in
accordance with the provisions described in Section 12.1.
5.6.2 Column 2: 180 cm long by 2 mm ID glass, packed with Ultrabond 20M (100/120 mesh)
or equivalent.
5.6.3 Detector: Electron capture. This detector has proven effective in the analysis of
wistewaters for the parameters listed in the scope and was used to develop the method
performance statements in Section 15. Alternative detectors, including a mass
spectrometer, may be used in accordance with the provisions described in Section 12.1.
6. REAGENTS
6.1 Reagent water: Reagent water is defined as a water in which an interferent is not observed at the
method detection limit of each parameter of interest.
6.2 Acetone, hexane, methylene chloride: Pesticide-quality or equivalent.
6.3 Sodium sulf e: ACS granular, anhydrous. Condition by heat in a shallow tray at 400°C for a
minimum of 4 hours to remove phthalates and other interfering organic substances. Alternatively,
heat I6ho45OtoS OO°Cinasha llowtrayorperfromaSoxhletextractionwithmethylene
chloride 1kw 48 hours.
6.4 Stock standard solutions (1.00 g/ LL): Stock standard solutions may be prepared from pure
s*”Iard materials or purchased as certified solutions.
6.4.1 Prepare stock standard solutions by accurately weighing approximately 0.0100 g of pure
material. Dissolve the material in pesticide-quality hexane and dilute to volume in a
1O-mL volumetric flask. Larger volumes may be used at the convenience of the analyst.
If compound purity is certified at 96% or greater, the weight may be used without
correction to calculate the concentration of the stock standard. Commercially prepared
stock standards may be used at any concentration if they are certified by the manufacturer
or by an independent source.
6.4.2 Transfer the stock standard solutions into TFE-fluorocarbon-sealed screw-cap vials.
Store at 4°C and protect from light Frequently check stock standard solutions for signs
of degradation or evaporation, especially just prior to preparing calibration standards
from them.
274
-------
Method 627
6.4.3 Stock standard solutions must be replaced after 6 months, or sooner if comparison with
check standards indicates a problem.
7. CALIBRA TION
7.1 Establish gas chromatographic operating parameters equivalent to those indicated in Table 1.
These parameters do not adequately resolve benfluralin, ethalfiuralin, and trifluralin. When more
than one of these compounds may be present in a sample, the instrument must be calibrated with
trifluralin. The gas chromatographic system may be calibrated using either the external standard
technique (Section 7.2) or the internal standard technique (Section 7.3).
7.2 External standard calibration procedure.
7.2.1 For each parameter of interest, prepare calibration standards at a minimum of three
concentration levels by adding accurately measured volumes of one or more stock
standards to a volumetric flask and diluting to volume with hexane. One of the external
standards should be representative of a concentration near, but above, the method
detection limit. The other concentrations should correspond to the range of
concentrations expected in the sample concentrates or should define the working range
of the detector.
7.2.2 Using injections of I to 5 giL of each calibration standard, tabulate peak height or area
responses against the mass injected. The results can be used to prepare a calibration
curve for each parameter. Alternatively, the ratio of the response to the mass injected,
defined as the calibration factor (CF), may be calculated for each parameter at each
standard concentration. If the relative standard deviation of the calibration factor is less
than 10% over the working range, the average calibration factor can be used in place of
a calibration curve.
7.2.3 The working calibration curve or calibration factor must be verified on each working
shift by the measurement of one or more calibration standards. If the response for any
parameter varies from the predicted response by more than ± 10%, the test must be
repeated using a fresh calibration standard. Alternatively, a new calibration curve or
calibration factor must be prepared for that parameter.
7.3 Internal standard calibration procedure: To use this approach, the analyst must select one or more
internal standards similar in analytical behavior to the compounds of interest. The analyst must
further demonstrate that the measurement of the internal standard is not affected by method or
matrix interferences. Due to these limitations, no internal standard applicable to all samples can
be suggested.
7.3.1 Prepare calibration standards at a minimum of three concentration levels for each
parameter of interest by adding volumes of one or more stock standards to a volumetric
flask. To each calibration standard, add a known constant amount of one or more
internal standards, and dilute to volume with hexane. One of the standards should be
representative of a concentration near, but above, the method detection limit. The other
concentrations should correspond to the range of concentrations expected in the sample
concentrates, or should define the working range of the detector.
275
-------
Method 627
7.3.2 Using injections of I to 5 ,iL of each calibration standard, tabulate the peak height or
area responses against the concentration for each compound and internal standard.
Calculate response factors (RF) for each compound as follows:
Equation 1
= ( A,)(C,, )
(Au) (C,)
y.*ere
A, = Response for the parameter to be measured
4, = Response for the intenzal standard
= Concentration of the Internal standard, in ig/L
C, Concentration of the parameter to be measured, in gIL
If the RF value over the working range is constant, less than 10% relative standard
deviation, the RF can be assumed to be invariant and the average RF may be used for
calculations. Alternatively, the results may be used to plot a calibration curve of
response ratios, AJA against RF.
7.3.3 The working calibration curve or RF must be verified on each working shift by the
measurement of one or more calibration standards. If the response for any parameter
varies from the predicted response by more than ± 10%, the test must be repeated using
a fresh calibration standard. Alternatively, a new calibration curve must be prepared for
that compound.
7.4 Before using any cleanup procedure, the analyst must process a series of calibration standards
through the procedure to validate elution patterns and the absence of interference from the
reagents.
8. Qu41.nv CONTROL
8.1 Each laboratory using this method is required to operate a formal quality control program. The
minimum requirements of this program consist of an initial demonstration of laboratory capability
and the analysis of spiked samples as a continuing check on performance. The laboratory is
required to m2intain performance records to define the quality of data that is generated.
8.1.1 Before performing any analyses, the analyst must demonstrate the ability to generate
acceptable accuracy and precision with this method. This ability is established as
described in Section 8.2.
8.1.2 In recognition of the rapid advances occurring in chromatography, the analyst is
permitted certain options to improve the separations or lower the cost of measurements.
E ith time such modifications to the method are made, the analyst is required to repeat
the procedure in Section 8.2.
8.1.3 The laboratory must spike and analyze a minimum of 10% of all samples to monitor
continuing laboratory performance. This procedure is described in Section 8.4.
276
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Method 627
8.2 To establish the ability to generate acceptable accuracy and precision, the analyst must perform
the following operations.
8.2.1 Select a representative spike concentration for each compound to be measured. Using
stock standards, prepare a quality control check sample concentrate in acetone, 1000
times more concentrated than the selected concentrations.
8.2.2 Using a pipette, add 1.00 mL of the check sample concentrate to each of a minimum of
four 1000-mL aliquots of reagent water. A representative wastewater may be used in
place of the reagent water, but one or more additional aliquots must be analyzed to
determine background levels, and the spike level must exceed twice the background level
for the test to be valid. Analyze the aliquots according to the method beginning in
Section 10.
8.2.3 Calculate the average percent recovery (R), and the standard deviation of the percent
recovery (s), for the results. Wastewater background corrections must be made before
R and s calculations are performed.
8.2.4 Table 2 provides single-operator recovery and precision for isopropalin, profiuralin and
trifluralin. Similar results should be expected for benfiuralin and ethalfiuralin. Compare
these results to the values calculated in Section 8.2.3.
8.3 The analyst must calculate method performance criteria and define the performance of the
laboratory for each spike concentration and parameter being measured.
8.3.1 Calculate upper and lower control limits for method performance as follows:
Upper Control Limit (UCL) = R + 3s
Lower Control Limit (LCL) = R - 3s
where R and s are calculated as in Section 8.2.3. The UCL and LCL can be used to
construct control charts 8 that are useful in observing trends in performance.
8.3.2 The laboratory must develop and maintain separate accuracy statements of laboratory
performance for wastewater samples. An accuracy statement for the method is defined
as R ± s. The accuracy statement should be developed by the analysis of four aliquots
of wastewater as described in Section 8.2.2, followed by the calculation of R and s.
Alternatively, the analyst may use four wastewater data points gathered through the
requirement fur continuing quality control in Section 8.4. The accuracy statements
should be updated regularly. 8
8.4 The laboratory is required to collect in duplicate a portion of their samples to monitor spike
recoveries. The frequency of spiked sample analysis must be at least 10% of all samples or one
spiked sample per month, whichever is greater. One aliquot of the sample must be spiked and
analyzed as described in Section 8.2. If the recovery for a particular parameter does not fall
within the control limits for method performance, the results reported for that parameter in all
samples processed as part of the same set must be qualified as described in Section 13.3. The
laboratory should monitor the frequency of data so qualified to ensure that it remains at or
below 5%.
277
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Met!w’id 627
8.5 Before processing any samples, the analyst must demonstrate through the analysis of a l-L aliquot
of reagent water that all glassware and reagent interferences are under control. Each time a set
of samples is extracted or there is a change in reagents, a laboratory reagent blank must be
processed as a safeguard against laboratory contamination.
8.6 It is recommended that the laboratory adopt additional quality assurance practices for use with this
method. The specific practices that are most productive depend upon the needs of the laboratory
and the nature of the samples. Field duplicates may be analyzed to monitor the precision of the
sampling technique. When doubt exists over the identification of a peak on the chromatogram,
confirmatory techniques such as gas chromatography with a dissimilar column, specific element
detector, or mass spectrometer must be used. Whenever possible, the laboratory should perform
analysis of quality control materials and participate in relevant performance evaluation studies.
9. SAMPLE COLLECTiON, PRESERVA lION, AND HANDLING
9.1 Grab samples must be collected in glass containers. Conventional sampling practices 9 should be
followed; however, the bottle must not be prerinsed with sample before collection. Composite
samples should be collected in refrigerated glass containers in accordance with the requirements
of the progr*m. Automatic sampling equipment must be as free as possible of plastic and other
potential sources of contamination.
9.2 The samples must be iced or refrigerated at 4°C from the time of collection until extraction.
9.3 All samples must be extracted within 7 days and completely analyzed within 40 days of extraction.
10. SAMPLE EXTRACTiON
10.1 Mark the water meniscus on the side of the sample bottle for later determination of sample
volume. Pour the entire sample into a 2-L separatory fimnel.
10.2 Add 60 mL methylene chloride to the sample bottle, seal, and shake 30 seconds to rinse the inner
walls. Transfer the solvent to the separatory funnel and extract the sample by shaking the funnel
for 2 minutes with periodic venting to release excess pressure. Allow the organic layer to separate
from the water phase for a minimum of 10 minutes. If the emulsion interface between layers is
more than one-third the volume of the solvent layer, the analyst must employ mechanical
techniques to complete the phase separation. The optimum technique depends upon the sample,
but may Include stirring, filtration of the emulsion through glass wool, centrifugation, or other
physical methods. Collect the methylene chloride extract in a 250-mL Erlenmeyer flask.
10.3 Add a second 60-mL volume of methylene chloride to the sample bottle and repeat the extraction
procedure a second time, combining the extracts in the Erlenmeyer flask. Perform a third
extraction in the same manner.
10.4 Assemble a Kuderna-Danish (K-D) concentrator by attaching a 10-mL concentrator tube to a
500-mL evaporative flask. Other concentration devices or techniques may be used in place of the
K-D if the requirements of Section 8.2 are met.
10.5 Pour the combined extract through a drying column containing about 10 cm of anhydrous sodium
sulfate, and collect the extract in the K-D concentrator. Rinse the Erlenmeyer flask and column
with 20 to 30 mL of methylene chloride to complete the quantitative transfer.
278
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Method 627
10.6 Add one or two clean boiling chips to the evaporative flask and attach a three-ball Snyder column.
Prewet the Snyder column by adding about 1 mL methylene chloride to the top. Place the K-D
apparatus on a hot water bath, 60 to 65°C, so that the concentrator tube is partially immersed in
the hot water, and the entire lower rounded surface of the flask is bathed with hot vapor. Adjust
the vertical position of the apparatus and the water temperature as required to complete the
concentration in 15 to 20 minutes. At the proper rate of distillation, the balls of the column will
actively chatter but the chambers will not flood with condensed solvent. When the apparent
volume of liquid reaches 1 mL, remove the K-D apparatus and allow it to drain and cool for at
least 10 minutes.
10.7 Increase the temperature of the hot water bath to about 80°C. Momentarily remove the Snyder
column, add 50 mL of hexane and a new boiling chip, and reattach the Snyder column. Pour
about 1 mL of hexane into the top of the Snyder column and concentrate the solvent extract as
before. Elapsed time of concentration should be 5 to 10 minutes. When the apparent volume of
liquid reaches I mL, remove the K-D apparatus and allow it to drain and cool for at least 10
minutes.
10.8 Remove the Snyder column and rinse the flask and its lower joint into the concentrator tube with
1 to 2 mL of hexane and adjust the volume to 10 mL. A 5-mL syringe is recommended for this
operation. Stopper the concentrator tube and store refrigerated if further processing will not be
performed immediately. If the extracts will be stored longer than two days, they should be
transferred to TFE-fluorocarbon-sealed screw-cap vials. If the sample extract requires no further
cleanup, proceed with gas chromatographic analysis. If the sample requires cleanup, proceed to
Section 11.
10.9 Determine the original sample volume by refilling the sample bottle to the mark and transferring
the water to a l000-mL graduated cylinder. Record the sample volume to the nearest 5 mL.
11. CLEANUP AND SEPARA TION
11.1 Cleanup procedures may not be necessary for a relatively clean sample matrix. If particular
circumstances demand the use of a cleanup procedure, the analyst must determine the elution
profile and demonstrate that the recovery of each compound of interest for the cleanup procedure
is no less than 85%.
12. GAS CHROMATOGRAPHY
12.1 Table 1 summarizes the recommended operating conditions for the gas chromatograph. Included
in this table are estimated retention times and method detection limits that can be achieved by this
method. An example of the separations achieved by Column 1 is shown in Figure 1. Other
packed columns, chromatographic conditions, or detectors may be used if the requirements of
Section 8.2 are met. Capillary (open-tubular) columns may also be used if the relative standard
deviations of responses for replicate injections are demonstrated to be less than 6% and the
requirements of Section 8.2 are met.
12.2 Calibrate the system daily as described in Section 7. Since the gas chromatographic conditions
provided do not adequately separate benfluralin, ethalfiuralin, and trifluralin, calibrate with
trifluralin if more than one of these materials may be present in a sample.
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Method 627
12.3 If the internal standard approach is being used, add the internal standard to sample extracts
immediately before injection into the instrument. Mix thoroughly.
12.4 Inject I to 5 pL of the sample extract using the solvent-flush technique.’° Record the volume
injected to the nearest 0.05 giL, and the resulting peak size in area or peak height units. An
automated system that consistently injects a constant volume of extract may also be used.
12.5 The width of the retention-time window used to make identifications should be based upon
measurements of actual retention-time variations of standards over the course of a day. Three
times the standard deviation of a retention time can be used to calculate a suggested window size
for a compound. However, the experience of the analyst should weigh heavily in the
Interpretation of chromatograms.
12.6 If the response for the peak exceeds the working range of the system, dilute the extract and
reanalyze.
12.7 If the measurement of the peak response is prevented by the presence of interferences, further
cleanup is required.
13. CALCULA7 ONS
13.1 Determine the concentration of individual compounds in the sample.
13.1.1 If the external standard calibration procedure is used, calculate the amount of material
injected from the peak response using the calibration curve or calibration factor in
Section 7.2.2. The concentration in the sample can be calculated as follows:
Equation 2
( A)(V )
Concentration, &gIL = (V,XV,)
A = Amount of material infected, in ng
V 1 Volume of extract injected, in p.L
V 1 = Volume of total extract, in pL
Volume of wzter extracted, in niL
13.1.2 If the internal standard calibration procedure was used, calculate the concentration in the
sample using the response factor (RF) determined in Section 7.3.2 as follows:
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Method 627
Equation 3
Concentration, ig/L = (A )(RF)(V 0 )
where
A, = Response for parameter to be measured
A = Response for the internal standard
I , = Amount of internal standard added to each extract,
in
zg
V 0 = Volume of water extracted, in L
13.2 Report results in micrograms per liter without correction for recovery data. When duplicate and
spiked samples are analyzed, report all data obtained with the sample results. Results for
benfluralin and ethalfiuralin must be reported as trifluralin unless the sample has been
characterized beyond the capabilities provided in this method.
13.3 For samples processed as part of a set where the laboratory spiked sample recovery falls, outside
of the control limits in Section 83, data for the affected parameters must be labeled as suspect.
14. GC/MS CONFIRMA T1ON
14.1 It is recommended that GCIMS techniques be judiciously employed to support qualitative
compound identifications made with this method. The mass spectrometer should be capable of
scanning the mass range from 35 amu to a mass 50 amu above the molecular weight of the
compound. The instrument must be capable of scanning the mass range at a rate to produce at
least 5 scans per peak but not to exceed 7 seconds per scan utilizing a 70 V (nominal) electron
energy in the electron impact ionization mode. A UC to MS interface constructed of all glass or
glass-lined materials is recommended. A computer system should be interfaced to the mass
spectrometer that allows the continuous acquisition and storage on machine-readable media of all
mass spectra obtained throughout the duration of the chromatographic program.
14.2 Gas chromatographic columns and conditions should be selected for optimum separation and
performance. The conditions selected must be compatible with standard GC/MS operating
practices. Chromatographic tailing factors of less than 5.0 must be achieved.’ 1
14.3 At the begimiing of each day that confirmatory analyses are to be performed, the GC/MS system
must be checked to see that all decafluorotriphenyl phosphine (DFTPP) performance criteria are
achieved.’ 2
14.4 To confirm an identification of a compound, the background-corrected mass spectrum of the
compound must be obtained from the sample extract and compared with a mass spectrum from
a stock or calibration standard analyzed under the same chromatographic conditions. It is
recommended that at least 25 ng of material be injected into the GCIMS. The criteria below must
be met for qualitative confirmation.
14.4.1 All ions that are present above 10% relative abundance in the mass spectrum of the
standard must be present in the mass spectrum of the sample with agreement to ±10%.
For example, if the relative abundance of an ion is 30% in the mass spectrum of the
standard, the allowable limits for the relative abundance of that ion in the mass spectrum
for the sample would be 20 to 40%.
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Method 627
14.4.2 The retention time of the compound in the sample must be within 6 seconds of the same
compound in the standard solution.
14.4.3 Compounds that have very similar m2s-c spectra can be explicitly identified by GCIMS
only on the basis of retention time data.
14.5 Where available, chemical ionization mass spectra may be employed to aid in the qualitative
identification process.
14.6 Should these MS procedures fail to provide satisfactory results, additional steps may be taken
before reanalysis. These may include the use of alternate packed or capillary GC columns or
additional cleanup (Section 11).
15. METhoD PERFORMANCE
15.1 The method detection limit (MDL) is defined as the mininqjm concentration of a substance that
can be measured and repofted with 99% confidence that the value is above zero. ’ The MDL
concentrations listed In Table I were obtained using reagent water. 1
15.2 In a single laboratory (West Cost Technical Services, Inc.) using reagent water and effluents from
pesticide manufacturers and the average recoveries presented in Table 2 were obtained. 1 The
standard deviations of the percent recoveries of these measurements are also included in Table 2.
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Method 627
References
1. “Pesticide Methods Evaluation,” Letter Report #5 for EPA Contract No. 68-03-2697. Available
from U.S. Environmental Protection Agency, Environmental Monitoring and Support Laboratory,
Cincinnati, Ohio.
2. ASTM Annual Book of Standards, Part 31, D3694, “Standard Practice for Preparation of Sample
Containers and for Preservation, U American Society for Testing and Materials, Philadelphia,
Pennsylvania, p. 679, 1980.
3. (3iam, D.S., Chan, H.S. and Nef, U.S., “Sensitive method for Determination of Phthalate Ester
Plasticizers in Open-Ocean Biota Samples,” Analytical Chemistry, 47, 2225, 1975.
4. (3iam, C.S., Chan, H.S., “Control of Blanks in the Analysis of Phthalates in Air and Ocean Biota
Samples,” National Bureau of Standards (U.S.), Special Publication 442, pp. 701-708, 1976.
5. “Carcinogens - Working with Carcinogens,” Department of Health, Education, and Welfare,
Public Health Service, Center for Disease Control, National Institute for Occupational Safety and
Health, Publication No. 77-206, Aug. 1977.
6. “OSHA Safety and Health Standards, General Industry,” (29 CFR 1910), Occupational Safety and
Health Administration, OSHA 2206, (Revised, January 1976).
7. “Safety in Academic Chemistry Laboratories,” American Chemical Society Publication,
Committee on Chemical Safety, 3rd Edition, 1979.
8. “Handbook for Analytical Quality Control in Water and Wastewater Laboratories,”
EPA-600/4.-79-019, U. S. Environmental Protection Agency, Environmental Monitoring and
Support Laboratory - Cincinnati, Ohio, March 1979.
9. ASTM Annual Book of Standards, Part 31, D3370, “Standard Practice for Sampling Water,”
American Society for Testing and Materials, Philadelphia, Pennsylvania, p. 76, 1980.
10. Burke, J.A., “Gas Chromatography ‘or Pesticide Residue Analysis; Some Practical Aspects,”
Journal of the Association of Official Analytical Chemists, 48, 1037, 1965.
11. McNair, H.M,, and Bonelli, E.J., Basic Chromatography, Consolidated Printing, Berkeley,
California, p. 52, 1969.
12. Eichelberger, J.W., Harris, L.E., and Budde, W.L. “Reference Compound to Calibrate Ion
Abundance Measurement in Gas Chromatography-Mass Spectrometry,” Analytical Chemistry, 47,
995, 1975.
13. Glaser, J.A. et.al, “Trace Analysis for Wastewaters,” Environmental Science & Technology, 15,
1426, 1981.
283
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Method 627
Table 1. Gas Chromatography and Method Detection Limits of Dinitroanhlines
Retention Time Minimum Method
Detection Limit
Column 1 Column 2
Parameter (pg/Li
Trifluralin 1.6 2.2 0.03
Benfluralin 1.6 2.3 ND
Eth n ifluralin 1.6 2.3 ND
Profluralin 2.3 3.4 0.14
Iscpropalin 6.4 6.3 0.02
ND = Not determined.
Column I conditions: Gas Chrom Q (100/200 mesh) coated with 1.5% OV-17I1.95% OV-210 packed
ma 1 .8m longby 2mm lDglassco lumnwith95% argon/5% methane Carriergas ataflowrateof 30
mL/inin. Colump temperature: isothermal at 190°C.
Column 2 conditions: Ultrabond 20M (100/120 mesh) packed in a 1.8 m long by 2 mm ID glass column
with nitrogen carrier gas at a flow rate of 30 mL/min. Column temperature: held at 160°C for 2
minutes, then programmed to 200°C at 10°C/mm.
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Method 627
Table 2. Single-Operator Accuracy and Precision
Average Standard
Sample Spike Range Number of Percent Deviation
Parameter Type (pg/LI Replicates Recovery %
Benfluralin 1W 2.00 2 93 —
Isopropalin DW 0.50 7 93 1.1
1W 2.20 7 88 13.2
Profluralin DW 0.50 7 99 9.0
1W 2.04 7 73 5.8
Trifluralin DW 0.50 7 97 1.8
1W 2.08 7 77 20.0
1W = Industrial wastewater, pesticide manufacturing
DW = Reagent water
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Mthrjd 627
0
1 I I I I I I I
1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0
Reterdion Tkn (minutes)
Figure 1. Gas Chromatogram of Dinitroaniline Pesticides on Column 1.
For Conditions, See Table 1.
A52 O .5B
Triflurain
Profkjrafr.
/
/opmpakn
a6
-------
Method 629
The Determination of Cyanazine
in Municipal and Industrial
Waste water
-------
Method 629
The Determination of Cyanazine in Municipal and Industrial
Wastewater
1. SCOPE AND APPL1CA T1ON
1.1 This method covers the determination of cyanazine. The following parameter can be determined
by this method:
Parameter STORET NO. CAS No.
Cyanazine 21725-46-2
1.2 This is a high performance liquid chromatographic (HLPC) method applicable to the determination
of the compound listed above in industrial and municipal discharges as provided under 40 CFR
136.1. Any modification of this method beyond those expressly permitted shall be considered a
major modification subject to application and approval of alternative test procedures under4O CFR
136.4 and 136.5.
1.3 The estimated method detection limit (MDL, defined in Section 15) for cyanazine is 6 ,tg/L. The
MDL for a specific wastewater may differ from those listed, depending upon the nature of inter-
ferences in the sample matrix.
1.4 This method is restricted to use by or under the supervision of analysts experienced in the use of
liquid chromatography and in the interpretation of liquid chromatograms. Each analyst must
demonstrate the ability to generate acceptable results with this method using the procedure
described in Section 8.2.
1.5 When this method is used to analyze unfamiliar samples for cyanazine, compound identifications
should be supported by at least one additional qualitative technique.
2. SUMMARY OF METHOD
2.1 A measured volume of sample, approximately 1 L, is extracted with methylene chloride using a
separatory funnel. The methylene chloride extract is dried and exchanged to methanol during
concentration to a volume of 10 mL or less. HPLC conditions are described which permit the
separation and measurement of cyanazine in the extract by HPLC with a UV detector. 1
2.2 This method provides an optional Florisil column cleanup procedure to aid in the elimination or
reduction of interferences which may be encountered.
3. INTERFERENCES
3.1 Method interferences may be caused by contaminants in solvents, reagents, glassware, and other
sample-processing apparatus that lead to discrete artifacts or elevated baselines in gas
chromatograms. All reagents and apparatus must be routinely demonstrated to be free from
interferences under the conditions of the analysis by running laboratory reagent blanks as
described in Section 8.5.
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Method 629
3.1.1 Glassware must be scrupulously cleaned. 2 Clean all glassware as soon as possible after
use by thoroughly rinsing with the last solvent used in it. Follow by washing with hot
water and detergent and thorough rinsing with tap and reagent water. Drain dry, and heat
in an oven or muffle furnace at 400°C for 15 to 30 minutes. Do not heat volumetric
ware. Thermally stable materials, such as PCBs, may not be eliminated by this
treatment. Thorough rinsing with acetone and pesticide-quality hexane may be substituted
for the heating. After drying and cooling, seal and store glassware in a clean
environment to prevent any accumulation of dust or other contaminants. Store inverted
or capped with aluminum foil.
3.1.2 The use of high-purity reagents and solvents helps to minimize interference problems.
Purification of solvents by distillation in all-glass systems may be required.
3.2 Matrix interferences may be caused by contaminants that are coextracted from the sample. The
extent of matrix interferences will vary considerably from source to source, depending upon the
nature and diversity of the industrial complex or municipality sampled. The cleanup procedure
In SectIon 11 can be used to overcome many of these interferences, but unique samples may
require additional cleanup approaches to achieve the MDL listed in Table 1.
4. SAFETY
4.1 The toxicity or carcinogenicity of each reagent used in this method has not been precisely defined;
however, each chemical compound must be treated as a potential health hazard. From this
viewpoint, exposure to these chemicals must be reduced to the lowest possible level by whatever
means available. The laboratory is responsible for maintaining a current awareness file of OSHA
regulations regarding the safe hawlling of the chemicals specified in this method. A reference file
of material data handling sheets should also be made available to all personnel involved in the
chemical analysis. Additional references to laboratory safety are available and have been
kIes*ifled 5 for the information of the analyst.
5. ArnRA TUS AND MA TEFJ4LS
5.1 Sampling equipment, for discrete or composite sampling.
5.1.1 Grab-sample bottle: Amber borosilicate or flint glass, 1-L or 1-quart volume, fitted with
screw-caps lined with TFE-fluorocarbon. Aluminum foil may be substituted for TFE if
the sample is not corrosive. If amber bottles are not available, protect samples from
lIght. The container and cap liner must be washed, rinsed with acetone or methylene
chloride, and dried before use to minimiie contamination.
5.1.2 Automatic sampler (optional): Must incorporate glass sample containers for the
collection of a mlninmm of 250 mL. Sample containers must be kept refrigerated at 4°C
and protected from light during compositing. If the sampler uses a peristaltic pump, a
mmiuuim length of compressible silicone rubber tubing may be used. Before use,
however, the compressible tubing must be thoroughly rinsed with methanol, followed by
repeated rirninga with reagent water to minimbe the potential for contamination of the
sample. An integrating flow meter is required to collect flow-proportional composites.
5.2 Glassware. (All specifications are suggested. Catalog numbers are included for illustration only.)
290
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Method 629
5.2.1 Separatory funnel: 2000 -mL, with TFE-fluorocarbon stopcock, ground-glass or TFE
stopper.
5.2.2 Drying column: Chromatographic column 400 mm long by 19 mm ID with coarse-fitted
disc.
5.2.3 Chromatographic column: 400 mm long by 19 mm ID with coarse-fitted disc at bottom
and TFE-fluorocarbon stopcock (Kontes K-420540-0224 or equivalent).
5.2.4 Concentrator tube, Kuderna-Danish: 10-mL, graduated (Kontes K-570050-1025 or
equivalent). Calibration must be checked at the volumes employed in the test. Ground-
glass stopper is used to prevent evaporation of extracts.
5.2.5 Evaporative flask, Kuderna-Danish: 500-mL (Kontes K-570001-0500 or equivalent).
Attach to concentrator tube with springs.
5.2.6 Snyder column, Kuderna-Danish: Three-baIl macro (Kontes K-503000-0121 or
equivalent).
5.2.7 Vials: Amber glass, 10- to 15-mL capacity with TFE-fluorocarbon-lined screw-cap.
5.3 Boiling chips: Approximately 10/40 mesh. Heat at 400°C for 30 minutes or perform a Soxhiet
extraction with methylene chloride.
5.4 Water bath: Heated, with concentric ring cover, capable of temperature control (±2°C). The
bath should be used in a hood.
5.5 Balance: Analytical, capable of accurately weighing to the nearest 0.0001 g.
5.6 Filtration apparatus: As needed to filter chromatographic solvents prior to HPLC.
5.7 Liquid chromatograph: High performance analytical system complete with high pressure syringes
or sample injection loop, analytical columns, detector, and strip-chart recorder. A guard column
is recommended for all applications.
5.7.1 Gradient pumping system, constant flow.
5.7.2 Column: 25 cm long by 2.6 mm ID stainless steel packed with Spherisorb ODS (10 &m)
or equivalent. This column was used to develop the method performance statements in
Section 14. Alternative columns may be used in accordance with the provisions
described in Section 12.1.
5.7.3 Detector: Ultraviolet, 254 nm. This detector has proven effective in the analysis of
wastewaters for cyanazine and was used to develop the method performance statements
in Section 14. Alternative detectors may be used in accordance with the provisions
described in Section 12.1.
6. REAGENTS
6.1 Reagent water: Reagent water is defined as a water in which an interferent is not observed at the
method detection limit of each parameter of interest.
6.2 Acetone, hexane, methylene chloride: Pesticide-quality or equivalent.
291
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Method 629
6.3 Ethyl ether: Nanograde, redistilled in glass if necessary. Must be free of peroxides as indicated
by EM Quant test strips (available from Scientific Products Co. Cat. No. P1126-8, and other
suppliers). Procedures recommended for removal of peroxides are provided with the test strips.
After deanup, 20 mL ethyl alcohol preservative must be added to each liter of ether.
6.4 Methanol: HPLC/UV quality.
6.5 Sodium sulfate: ACS, granular, anhydrous. Condition by beating in a shallow tray at 400°C for
a minimum of 4 hours to remove phthalates and other interfering organic substances.
Alternatively, heat 16 hours at 450 to 500°C in a shallow tray or perform a Soxhlet extraction
with methylene chloride for 48 hours.
6.6 Florisll: PR grade (601100 mesh). Purchase activated at 675°C and store in dark in glass
container with ground-glass stopper or foil-lined screw-cap. Before use, activate each batch at
least 16 hours at 130°C in a foil-covered glass container.
6.7 Stock standard solution (1.00 1 ig/ 1 iL): A stock standard solution may be prepared from pure
standard material or purchased as a certified solution.
6.7.1 Prq,are a stock standard solution by accurately weighing approximately 0.0100 g of
cyanazine. Dissolve the material in UV quality methanol and dilute to volume in a
I0-mL volumetric flask. Larger volumes may be used at the convenience of the analyst.
If compound purity is certified at 96% or greater, the weight may be used without
correction to calculate the concentration of the stock standard. Commercially prepared
stock standards may be used at any concentration if they are certified by the manufacturer
or by an independent source.
6.7.2 Transfer the stock standard solution into a TFE-fluorocarbon-sealed screw-cap vial.
Store at 4°C and protect from light. Frequently check the stock standard solution for
signs of degradation or evaporation, especially just prior to preparing calibration
standards from it.
6.7.3 The stock standard solution must be replaced after 6 months, or sooner if comparison
with a check standard indicates a problem.
7. CAUIRAJYON
7.1 Establish HPLC operating parameters equivalent to those indicated in Table 1. The HPLC system
may be calibrated using either the external standard technique (Section 7.2) or the internal
standard technique (Section 7.3).
7.2 External standard calibration procedure:
7.2.1 Prq*re calibration standards at a minimum of three concentration levels by adding
accurately measured volumes of stock standard to volumetric flasks and diluting to
volume with methanol. One of the external standards should be representative of a
coecentration near, but above, the method detection limit. The other concentrations
should correspond to the range of concentrations expected in the sample concentrates or
should define the working range of the detector.
7.2.2 UsIng injections of 10 pgIL of each calibration standard , tabulate peak height or area
responses against the m s injected. The results can be used to prepare a calibration
curve for cyanazine. Alternatively, the ratio of the response to the mass injected, defined
292
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Method 629
as the calibration factor (CF), may be calculated at each standard concentration. If the
relative standard deviation of the calibration factor is less than 10% over the working
range, the average calibration factor can be used in place of a calibration curve.
7.2.3 The working calibration curve or calibration factor must be verified on each working
shift by the measurement of one or more calibration standards. If the response varies
from the predicted response by more than ± 10%, the test must be repeated using a fresh
calibration standard. Alternatively, a new calibration curve or calibration factor must be
prepared.
7.3 Internal standard calibration procedure: To use this approach, the analyst must select an internal
standard similar in analytical behavior to cyanazine. The analyst must further demonstrate that
the measurement of the internal standard is not affected by method or matrix interferences. Due
to these limitations, no internal standard applicable to all samples can be suggested.
7.3.1 Prepare calibration standards at a minimum of three concentration levels by adding
volumes of stock standard to volumetric flasks. To each calibration standard, add a
known constant amount of internal standard, and dilute to volume with methanol. One
of the standards should be representative of a concentration near, but above, the method
detection limit. The other concentrations should correspond to the range of
concentrations expected in the sample concentrates, or should define the working range
of the detector.
7.3.2 Using injections of 10 g/L of each calibration standard, tabulate the peak height or area
responses against the concentration for both cyanazine and internal standard. Calculate
response factors (RF) as follows:
Equation 1
RE = ( A,)(C, )
(A )(C ,)
where
A, = Response for the parameter to be measured
4 , = Response for the internal standard
C i = Concentration of the internal standard, in gIL
C, = Concentration of the parameter to be measured, in g/L
If the RF value over the working range is constant, less than 10% relative standard
deviation, the RF can be assumed to be invariant and the average RF may be used for
calculations. Alternatively, the results may be used to plot a calibration curve of
response ratios, A /A 1 against RF.
7.3.3 The working calibration curve or RF must be verified on each working shift by the
measurement of one or more calibration standards. If the response varies from the
predicted response by more than ± 10%, the test must be repeated using a fresh
calibration standard. Alternatively, a new calibration curve must be prepared.
293
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Method 629
7.4 The cleanup procedure in Section 11 utilizes Florisil chromatography. Florisil from different
batches or sources may vary in adsorptive capacity. To standardize the amount of Florisil which
is used, the use of the lauric acid value is suggested. This procedure 6 determines the adsorption
from hexane solution of lauric acid, in milligrauis per gram of Florisil. The amount of Florisil
to be used for each column is calculated by dividing this factor into 110 and multiplying by 20 g.
7.5 Before using any cleanup procedure, the analyst must process a series of calibration standards
through the procedure to validate elution patterns and the absence of interference from the
reagents.
8. 0u4uTv ColvmoL
8.1 P- ch laboratory using this method is required to operate a formal quality control program. The
minimum requirements of this program consist of an initial demonstration of laboratory capability
and the analysis of spiked samples as a continuing check on performance. The laboratory is
required to m2int in performance records to define the quality of data that is generated.
8.1.1 Before performing any analyses, the analyst must demonstrate the ability to generate
acceptable accuracy and precision with this method. This ability is established as
described in Section 82.
8.1.2 In recognition of the rapid advances occurring in chromatography, the analyst is
permitted certain options to improve the separations or lower the cost of measurements.
Fec 1 time such modifications to the method are made, the analyst is required to repeat
the procedure in Section 8.2.
8.1.3 The laboratory imist spike and analyze a minimum of 10% of all samples to monitor
cotifinuing laboratory performance. This procedure is described in Section 8.4.
8.2 To establish the ability to generate acceptable accuracy and precision, the analyst must perform
the following operations.
8.2.1 Select a representative spike concentration. Using stock standard, prepare a quality
control check sample concentrate in methanol, 1000 times more concentrated than the
selected concentrations.
8.2.2 Using a pipette, add 1.00 mL of the check sample concentrate to each of a minimum of
four 1000-mL aliquots of reagent water. A representative wastewater may be used in
place of the reagent water, but one or moró additional aliquots must be analyzed to
determine background levels; and the spike level must exceed twice the background level
for the test to be valid. Analyze the aliquots according to the method beginning in
SectIon 10.
8.2.3 Calculate the average percent recovery (R), and the standard deviation of the percent
recovery (a), for the results. Wastewater background corrections must be made before
R and $ calculations are performed.
8.2.4 Using the appropriate data from Table 2, determine the recovery and single-operator
precision expected for the method, and compare these results to the values calculated in
Section 8.2.3. If the data are not comparable, review potential problem areas and repeat
the test.
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Method 629
8.3 The analyst must calculate method performance criteria and define the performance of the
laboratory for each spike concentration being measured.
83.1 Calculate upper and lower control lunits for method performance as follows:
Upper Control Limit (UCL) = R + 3s
Lower Control Limit (LCL) = R - 3s
where R and s are calculated as in Section 8.2.3. The UCL and LCL can be used to
construct control charts 7 that are useful in observing trends in performance.
8.3.2 The laboratory must develop and maintain separate accuracy statements of laboratory
performance for wastewater samples. An accuracy statement for the method is defined
as R ± s. The accuracy statement should be developed by the analysis of four aliquots
of wastewater as described in Section 8.2.2, followed by the calculation of R and s.
Alternatively, the analyst may use foUr wastewater data points gathered through the
requirement for continuing quality control in Section 8.4. The accuracy statements
should be updated regularly. 7
8.4 The laboratory is required to collect in duplicate a portion of their samples to monitor spike
recoveries. The frequency of spiked sample analysis must be at least 10% of all samples or one
spiked sample per month, whichever is greater. One aliquot of the sample must be spiked and
analyzed as described in Section 8.2. If the recovery of cyanazine does not fall within the control
limits for method performance, the results reported for cyanazine in all samples processed as part
of the same set must be qualified as described in Section 13.3. The laboratory should monitor
the frequency of data so qualified to ensure that it remains at or below 5%.
8.5 Before processing any samples, the analyst must demonstrate through the analysis of a 1-L aliquot
of reagent water that all glassware and reagent interferences are under control. Each time a set
of samples is extracted or there is a change in reagents, a laboratory reagent blank must be
processed as a safeguard against laboratory contamination.
8.6 It is recommended that the laboratory adopt additional quality assurance practices for use with this
method. The specific practices that are most productive depend upon the needs of the laboratory
and the nature of the samples. Field duplicates may be analyzed to monitor the precision of the
sampling technique. When doubt exists over the identification of a peak on the chromatogram as
cyanazine, confirmatory techniques, such as chromatography with a dissimilar column or ratio of
absorbance at two or more wavelengths, must be used. Whenever possible, the laboratory should
perform analysis of quality control materials and participate in relevant performance evaluation
studies.
9. SAMPLE CollEcTioN, PRESERVATiON, AND HANDLING
9.1 Grab samples must be collected In glass containers. Conventional sampling practices 8 should be
followed; however, the bottle must not be prerinsed with sample before collection. Composite
samples should be collected in refrigerated glass containers in accordance with the requirements
of the program. Automatic sampling equipment must be as free as possible of plastic and other
potential sources of contamination.
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Method 629
9.2 The samples must be iced or refrigerated at 4°C from the time of collection until extraction.
9.3 All samples must be extracted within 7 days and completely analyzed within 40 days of extraction.
10. SAMPLE ExmAclloN
10.1 Mark the water meniscus on the side of the sample bottle for later determination of sample
volume. Pour the entire sample into a 2 -L separatory funnel.
10.2 Add 60 mL inethylene chloride to the sample bottle, seal, and shake 30 seconds to rinse the inner
walls. Transfer the solvent to the separatory funnel and extract the sample by shaking the funnel
for 2 minutes with periodic venting to release excess pressure. Allow the organic layer to separate
from the water phase for a minimum of 10 minutes. If the emulsion interface between layers is
more than one-third the volume of the solvent layer, the analyst must employ mechanical
techniques to complete the phase separation. The optimum technique depends upon the sample,
but may include stirring, filtration of the emulsion through glass wool, centrifugation, or other
physical methods. Collect the methylene chloride extract in a 250-mL Erlenmeyer flask.
10.3 Add a second 60-mL volume of methylene chloride to the sample bottle and repeat the extraction
procedure a second time, combining the extracts in the Erlenmeyer flask. Perform a third
extraction in the same manner.
10.4 ItlsnecessarytoexthangetheextractSolventtohexaneiftheF10risilCleaflUPP1OCedUre t0be
used. For direct HPLC analysis, the extract solvent must be changed to methanol. The analyst
should only exchange a portion of the extract to methanol if there is a possibility that cleanup may
be necessary.
10.5 Assemble a Kuderna-Danish (K-I)) concentrator by altat 4 ting a 10-mL concentrator tube to a
500-mL evaporative flask. Other concentration devices or techniques may be used in place of the
IC-D if the requirements of Section 8.2 are met.
10.6 POur a measured fraction or all of the combined extract through a drying column conbining about
10 cm of anhydrous sodium sulfate, and collect the extract in the K-D concentrator. Rinse the
Erlenmeyer flask and column with 20 to 30 mL of methylene chloride to complete the quantitative
transfer.
10.7 Add I or 2 clean boiling chips to the evaporative flask and attach a three-ball Snyder column.
Prewet the Snyder column by adding about 1 mL methylene chloride to the top. Place the K-D
apparatus on a hot water bath, 60 to 65°C, so that the concentrator tube is partially immersed in
the hot water, and the entire lower rounded surface of the flask is bathed with hot vapor. Adjust
the vestical position of the apparatus and the water temperature as required to complete the
concentrationin 15 to 20 minutes. Attheproper rate ofdistillation,theballsofthe column will
actively ch ter but the chambers will not flood with condensed solvent. When the apparent
volume of liquid reaches 1 mL, remove the lCD apparatus and allow it to drain and cool for at
leastlOmin. -
10.8 Increase the temperature of the hot water bath to about 80°C. Momentarily remove the Snyder
column, add 50 mL of hexane or methanol and a new boiling chip, and reattach the Snyder
column. Pour about 1 mL of solvent into the top of the Snyder column and concentrate the
solvent extract as before. Elapsed time of concentration should be 5 to 10 minutes. When the
296
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Method 629
apparent volume of liquid reaches 1 mL, remove the K-D apparatus and allow it to drain and cool
for at least 10 minutes.
10.9 Remove the Snyder column and rinse the flask and its lower joint into the concentrator tube with
1 to 2 mL of hexane or methanol and adjust the volume to 10 mL. A 5-mL syringe is
recommended for this operation. Stopper the concentrator tube and store refrigerated if further
processing will not be performed immediately. If the extracts will be stored longer than 2 days,
they should be transferred to TFE-fluorocarbon-sealed screw-cap vials. If the sample extract
requires no further cleanup, proceed with HPLC analysis. If the sample requires cleanup, proceed
to Section 11.
10.lODetermine the original sample volume by refilling the sample bottle to the mark and transferring
the water to a 1000-mL graduated cylinder. Record the sample volume to the nearest 5 mL.
11. CLEANUP AND SEPARA TION
11.1 Cleanup procedures may not be necessary for a relatively clean sample matrix. The cleanup
procedure recommended in this method has been used for the analysis of various industrial and
municipal effluents. If particular circumstances demand the use of an alternative cleanup
procedure, the analyst must determine the elution profile and demonstrate that the recovery of
cyanazine for the cleanup procedure is no less than 85%.
11.2 The following Florisil column cleanup procedure has been demonstrated to be applicable to
cyanazine.
11.2.1 Add a weight of Florisil (nominally 20 g) predetermined by calibration (Section 7.4
and 7.5) to a chromatographic column. Settle the Florisil by tapping the column. Add
anhydrous sodium sulfate to the top of the Florisil to form a layer 1 to 2 cm deep. Add
60 mL of hexane to wet and rinse the sodium sulfute and Florisil. Just prior to exposure
of the sodium sulfute to air, stop the elution of the hexane by closing the stopcock on the
chromatography column. Discard the eluate.
11.2.2 Adjust the sample extract volume to 10 mL with hexane and transfer it from the K-D
concentrator tube to the Florisil column. Rinse the tube twice with 1 to 2 mL hexane,
adding each rinse to the column.
11.2.3 Drain the column until the sodium sulfate layer is nearly exposed. Elute the column with
200 mL of 6% ethyl ether in hexane (V/V) (Fraction 1) and with 200 mL of 15% ethyl
ether in hexane (VJV) (Fraction 2) using a drip rate of about 5 mL/min. These fractions
may be discarded. Place a 500-mL K-D flask and clean concentrator tube under the
chromatography column. Elute the column with 200 mL of 50% ethyl ether in hexane
(VIV) (Fraction 3) into the K-D flask. Cyanazine elutes quantitatively in Fraction 3.
11.2.4 Concentrate the eluate by standard K-D techniques (Section 10.7), exchanging the solvent
to methanol. Adjust final volume to 10 mL with methanol. Analyze by HPLC.
12. LIQuID CHROMATOGRAPHY
12.1 Table I summarizes the recommended operating conditions for the liquid chromatograph.
Included in this table are the estimated retention time and method detection limit that can be
achieved by this method. An example of the separations achieved by this column is shown in
297
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Method 629
Figure 1. Other HPLC columns, chromatoaraohic conditions, or detectors may be used if the
requirements of Section 8.2 are met.
12.2 Calibrate the system daily as described in Section 7.
12.3 If the internal standard approach is being used, add the internal standard to sample extracts
immediately before injection into the instrument. Mix thoroughly.
12.4 Inject 10 ig/L of the s2rl ,le extract. Record the volume injected to the nearest 0.05 pL, and the
resulting peak size in area or peak height units.
12.5 The width of the retention-time window used to make identifications should be based upon
measurements of actual retention-time variations of standards over the course of a day. Three
times the sisnd2rd deviation of a retention time can be used to calculate a suggested window size
for a compound. However, the experience of the analyst should weigh heavily in the
interpretation of chromatograms.
12.6 If the response for the peak exceeds the working range of the system, dilute the extract and
reanalyze.
12.7 If the measurement of the peak response is prevented by the presence of interferences, further
cbs is required.
13. CALCULATiONS
13.1 DetermIne the concentration of cyanazine in the sample.
13.1.1 If the external stsndprd calibration procedure is used, calculate the amount of material
injected from the peak response using the calibration curve or calibration factor in
Section 7.2.2. The concentration in the sample can be calculated as follows:
EquaUcn2
( I4XV )
Conce,uratlon, NIL =_____
A - Ansowit qr qaiswine bifected, hi nanograins.
v- Vo4J nse ojexhact Injected, In 1 agIL
V 1 Volwsse q total extract, in igIL
Vs - Valwae qf nuter extracted, hi niL
13.1.2 If the internal stsnd2rd calibration procedure was used, calculate the concentration in the
s’n 4e using the response factor (RF) determined in Section 7.3.2 as follows:
298
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Method 629
Equation 3
Concentration, igIL =
(A 1 )(RF)(V 0 )
where:
A, = Response for cyanazine
A = Response for the internal standard
1, = Amount of internal standard added to each
extract,
in
,ig
V 0 = Volume of water extracted, in liters
13.2 Report results in micrograms per liter without correction for recovery data. When duplicate and
spiked samples are analyzed, report all data obtained with the sample results.
13.3 For samples processed as part of a set where the laboratory spiked sample recovery falls outside
of the control limits in Section 8.3, data for cyanazine must be labeled as suspect.
14. METHOD PERFORMANCE
14.1 The method detection limit (MDL) is defined as the minimum concentration of a substance that
can be measured and reported with 99% confidence that thevalue is above zero. 9 The MDL
concentration listed in Table 1 was estimated from the response of a 254 nm UV detector to the
compound. The estimate is based upon the amount of material required to yield a signal 5 times
the HPLC background noise, assuming a 1O- ig injection from a 1O-mL final extract of a 1-L
sample.
14.2 In a single laboratory (West Cost Technical Services, Inc.), using effluents from pesticide
manufacturers and publicly owned treatment works (POTW), the average recoveries presented in
Table 2 were obtained.’ The standard deviations of the percent recoveries of these measurements
are also included in Table 2.
299
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Method 629
References
1. uPesticide Methods Evaluation, Letter Report for EPA Contract No. 68-03-2697. Available from
U.S. Environmental Protection Agency, Environmental Monitoring and Support Laboratory,
Cincinn i, Ohio.
2. ASTM Annual Book of Standards, Part 31, D3694, US J Practice for Preparation of Sample
Containers and for Pr ation U American Society for Testing and Materials, Philadelphia,
Pennsylvania, p. 679, 1980.
3. Carclnogens - Working with Carcinogens, Department of Health, Education, and Welfare,
Public Health Service, Center for Disease Control, National Institute for Occupational Safety and
Health, Publication No. 77-206, Aug. 1977.
4. OSHA Safety and Health Standards, General Industry, (29 CFR 1910), Occupational Safety and
Health Administration, OSHA 2206, (Revised, January 1976).
5. “Safety In Academic Chemistry Laboratories,” American Chemical Society Publication,
Committee on Chemical Safety, 3rd Edition, 1979.
6. ASTM Annual Book of Standards, Part 31, D3086, Appendix X3, “Standardization of Florisil
Column by Weight Adjustment Based on Adsorption of Lauric ACId,” American Society for
Testing and Materials, Philadelphia, PA, p. 765, 1980.
7. llandbook for Analytical Quality Control in Water and Wastewater Laboratories,”
EPA-600/4-79-019, U. S. Environmental Protection Agency, Environmental Monitoring and
Support Laboratory - Cincinnati, Ohio, March 1979.
8. ASTM Ani I Book of Standards, Part 31, D3370, “Standard Practice for Sampling Water,”
American Society for Testing and Materials, Philadelphia, PA, p. 76, 1980.
9. Glaser, J.A. elal, uTrace Analysis for Wastewaters,” Environmental Science & Technology, 15,
1426(1981).
-------
Method 629
Table 1. Chromatographic Conditions and Estimated Detection Limit
Retention Time Estimated MDL
Parameter (mini (pg/Li
Cyanazine 10.0 6
Column conditions: Spherisorb ODS (10 m) packed in a 25 cm long by 2.6 mm 11) stainless steel
column with a mobile phase flow rate of 1.0 mL/rnin. Mobile phase: Linear gradient from 50% solvent
B to 100% solvent B in 2 mm, where solvent A is 25% methanol in water and solvent B is 50% methanol
in water.
Table 2. Single-Operator Accuracy and Precision
Average Standard
Spike No. of Percent Deviation
Parameter Sample Type (pg/L) Replicates Recovery (%)
Cyanazine DW 121 7 100.0 8.9
MW 60.8 7 85.5 3.9
PW 10,100 3 94.3 -
1W 10,100 2 78.0
DW = Reagent water
MW = Municipal wastewater
PW = Process water, pesticide manufacturing
1W = Industrial wastewater, pesticide manufacturing
301
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Method 629
0 5.0 10.0
Figure 1. Liquid Chromatogram
Extract on Column 1.
ot Cyanazine in Process Water
For Conditions, See Table 1.
/ Cyanazine
Retention Time (minutes)
15.0
302
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Method 630
The Determination
of Dithiocarbama te
Pesticides in Municipal and
Industrial Waste water
-------
Method 630
The Determination of Dithiocarbamate Pesticides in Municipal and
Industrial Waste water
SCOPE AND APPLICATION
1.1 This method covers the determination of dithiocarbamate pesticides. The following parameters can
be determined by this method:
Parameter STORETNo. CAS. No.
Amoban 3566-10-7
AOP
Busan 40 51026-28-9
Busan 85 128-03-0
Ferbam 14484-64-1
KN Methyl 137-41-7
Mancozeb 8018-01-7
Maneb 12427-38-1
Metham 137-42-8
Nabam 142-59-6
Niacide 8011-66-3
Polyram 9006-42-2
Sodium dimethyldithiocarbaniate 128-04-1
Thiram 137-26-8
ZAC
Zineb 12122-67-7
Ziram 137-30-4
1.2 This method fails to distinguish between the individual dithiocarbamates. The compounds above
are reduced to carbon disulfide and the total dithiocarbamate concentration is measured. Unless
the sample can be otherwise characterized, all results are reported as Ziram. Carbon disulfide is
a known interferent.
1.3 This is a colorimetric method applicable to the determination of the compounds listed above in
industrial and municipal discharges as provided under 40 CFR 136.1. Any modification of this
method beyond those expressly permitted shall be considered a major modification subject to
application and approval of alternative test procedures under 40 CFR 136.4 and 136.5.
1.4 The method detection limit (MDL, defined in Section 12) for maneb, metham and ziram are listed
in Table 1. The MDL for a specific dithiocarbamate or wastewater may differ from those listed,
depending upon the nature of interferences in the sample matrix.
1.5 This method is restricted to use by or under the supervision of analysts experienced in trace
organic analyses. Each analyst must demonstrate the ability to generate acceptable results with
this method using the procedure described in Section 8.2.
305
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Method 630
2. SUMMARY OF METHOD
2.1 A measured volume of sample, approximately 1 L, is digested with acid to yield carbon disulfide
by hydrolysis of the dithiocarbamate moiety. The evolved CS 2 is purged from the sample and
absorbed by a color reagent. The absorbance of the solution is measured at 380 and 435 nrn using
a UV-visible spectrophotometer.’
3. INTERFERENCES
3.1 Method interferences may be caused by contaminants in reagents, glassware, and other sample
processing hardware that lead to high blank values and biased results. All of these materials must
be routinely demonstrated to be free from interferences under the conditions of the analysis by
running laboratory reagent blanks as described in Section 8.5.
3.1.1 Glassware must be scrupulously cleaned. 2 After each use, rinse the decomposition flask
and condenser with 4N NaOH and reagent water. Overnight soaking in 4N NaOH may
be necessary. Clean the H 2 S scrubber between each use with 0.IN HO in methanol,
rinse three times with methanol, and bake at 200°C for 15 minutes. Rinse the CS2 trap
with methanol three times between each use and follow by heating for 15 minutes at
200°C. Should it become difficult to force the color reagent through the glass fit of the
CS 2 trap, clean in the same manner as the 14 S scrubber. After cooling, store glassware
sealed to prevent any accumulation of dust or other contaminants.
3.1.2 The use of high-purity reagents and solvents helps to minimize interference problems.
3.2 Carbon disulfide may be a significant direct interferent in wastewaters. Its elimination or control
is not addressed in this method. If correction for background carbon disulfide is required, the CS 2
should be measured by an independent procedure, such as direct aqueous injection gas
chromatography.
3.3 Additional matrix interferences may be caused by contaminants that are codistilled from the
sample. The extent of matrix interferences will vary considerably from source to source,
depending upon the nature and diversity of the industrial complex or municipality being sampled.
The cleanup provided by the H 2 S trap will eliminate or reduce some of these interferences,
but unique samples may require additional clean-up approaches to achieve the MDL listed in
Table 1.
4. SAFETY
4.1 The toxicity or carcinogenicity of each reagent used in this method has not been precisely defined;
however, each chemical compound should be treated as a potential health hazard. From this view-
point, exposure to these chemicals must be reduced to the lowest possible level by whatever means
available. The laboratory is responsible for maintaining a current awareness file of OSHA
regulations regarding the safe handling of the chemicals specified in this method. A reference file
of material data handling sheets should also be made available to all personnel involved in the
chemical analysis. Additional references to laboratory safety are available and have been
identifled 5 for the information of the analyst.
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Method 630
5. APPARA TUS AND MA TERIALS.
5.1 Sampling equipment, for discrete or composite sampling.
5.1.1 Grab-sample bottle: Amber borosilicate or flint glass, 1-L or 1-quart volume, fitted with
screw-caps lined with TFE-fluorocarbon. Aluminum foil may be substituted for TFE if
the sample is not corrosive. If amber bottles are not available, protect samples from
light. The container and cap liner must be washed, rinsed with acetone or methylene
chloride, and dried before use to minimize contamination.
5.1.2 Automatic sampler (optional): Must incorporate glass sample containers for the
collection of a minimum of 250 mL. Sample containers must be kept refrigerated at 4°C
and protected from light during compositing. If the sampler uses a peristaltic pump, a
minimum length of compressible silicone rubber tubing may be used. Before use,
however, the compressible tubing must be thoroughly rinsed with methanol, followed by
repeated rinsings with reagent water to minimize the potential for contamination of the
sample. An integrating flow meter is required to collect flow-proportional composites.
5.2 Dithiocarbamate hydrolysis apparatus (Figure 1): (Available from Southern Scientific Inc., Box
83, Micanopy, Florida 32267). Apparatus includes the following or equivalent components:
5.2.1 Hot plate with magnetic stirrer.
5.2.2 Hydrolysis flask: 2-L, flat bottom with ground-glass joints, 2 necks.
5.2.3 Condenser: Low internal volume, ground-glass joints, Liebig (Kontes K-447000, 100
mm or equivalent).
5.2.4 Gas-washing bottles: 125-mL, with extra-coarse porosity (Kontes K-657750 or
equivalent).
5.2.5 Addition funnel: 60-mL, ground-glass joint to fit hydrolysis flask, with long stem to
reach at least 2 cm below the liquid level in the hydrolysis flask.
5.2.6 Dust trap (adapter): To fit top of addition funnel (Kontes K- 174000 or equivalent).
5.2.7 Vacuum source: Stable pressure with needle valve for control.
5.3 UV-visible spectrophotometer: Double beam with extended cell path length capability of 1.0 and
4.0 cm cells.
5.4 Balance: Analytical, capable of accurately weighing to the nearest 0.0001 g. The preparation of
calibration standards for some dithiocarbamates (e.g., metham) requires the use of a balance
capable of weighing 10 jig.
6. REAGENTS
6.1 Reagent water: Reagent water is defined as a water in which an interferent is not observed at the
method detection limit of each parameter of interest. !rePare by boiling distilled water 15 minutes
immediately before use.
6.2 Acetonitrile, diethanolamine, methanol: ACS grade.
6.3 Ethanol: 95%.
6.4 Cupric acetate: Monohydrate, ACS grade.
6.5 Hydrochloric acid: Concentrated.
307
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Method 630
6.6 Hydrochloric acid, O.1N in methanol: Slowly add 8.3 mL concentrated HCI to methanol and
dilute to 100 mL.
6.7 Sodium hydroxide, 4N: Dissolve 16 g ACS grade NaOH pellets in reagent water and dilute to
100 mL.
Stannous chloride: SnCI 2 • 2H 2 0, ACS grade.
Zinc acetate solution, 20%: Dissolve 20 g ACS grade Zn(C 2 H 3 0 2 ) 2H 2 0 in reagent water and
diluteto lOOmL.
6.10 Color reagent: Add 0.012 g cupric acetate monohydrate to 25 g diethanolamine. Mix thoroughly
while diluting to 250 mL with ethanol. Store in amber bottle with TFE-fluorocarbon-Iined cap.
6.11 Decomposition reagent: Dissolve 9.5 g stannous chloride in 300 mL concentrated hydrochloric
acid. Prepare fresh daily.
6.12 Stock standard solutions (1.00 g/ &L): Stock standard solutions may be prepared from pure
standard materials or purchased as certified solutions.
6.12.1
Prepare a stock standard solution for ziram by accurately weighing approximately
0.0100 g of pure material. Dissolve the material in acetonitrile and dilute to volume in
a 1-mL volumetric flask. Larger volumes may be used at the convenience of the analyst.
If compound purity is certified at 96% or greater, the weight may be used without
correction to calculate the concentration of the stock standard. Commercially prepared
stock standards may be used at any concentration if they are certified by the manufacturer
or by an independent source.
6.12.2 Transfer the stock standard solution into a TFE-fluorocarbon-sealed screw-cap vial.
Store at 4°C and protect from light. Frequently check stock standard solutions for signs
of degradation or evaporation, especially just prior to preparing calibration standards
from them.
61 2.3 Stock standard solutions must be replaced after 6 months, or sooner if comparison with
check standards indicates a problem.
6.12.4 When using other dithiocarbamates for calibration, such as maneb or metham, it may be
necessary to weigh microgram amounts of the pure material into small aluminum foil
boats and place them directly in the hydrolysis flask.
7. CAL/BRA T1ON
7.1 Use ziram as the standard for total dithiocarbamates when a mixture of dithiocarbamates is likely
to be present. Use the specific dithiocarbamate as a standard when only one pesticide is present
and its identity has been established.
7.2 With the apparatus assembled and reagents in place, Section 10, pour 1,500 mL of reagent water
into each decomposition flask, add 30 mL of decomposition reagent, and start aspiration.
7.3 Spike the water in each flask with an accurately known weight of dithiocarbamate standard. Use
a series of weights equivalent to 5 to 200 ig of CS 2 . Follow the procedure outlined Section 10.
7.4 Prepare calibration curves at a minimum of three concentrations by plotting absorbance vs. weight
of dithiocarbamate. A separate curve is prepared from readings taken at 435 nm and at 380 nm
for each cell path length used. Normally the 435 run curve is used for calibration above 30 g
6.8
6.9
308
-------
Method 630
ziram (4 cm cell), and the 380 nm curve is used tbr calibration below 30 g ziram. The choice
of which curve to use is left to the discretion of the analyst. It is recommended that the curves
be transformed into mathematical equations using linear least squares fit for the data from 435 nm
and quadratic least squares fit for data from the 380 nm.
7.5 The working calibration curve must be verified on each working shift by the measurement of one
or more calibration standards. If the response varies from the predicted response by more
than ±10%, the test must be repeated using a fresh calibration standard. Alternatively, a new
calibration curve must he prepared.
8. QUALITY CONTROL
8.1 Each laboratory using this method is required to operate a formal quality control program. The
minimum requirements of this program consist of an initial demonstration of laboratory capability
and the analysis of spiked samples as a continuing check on performance. The laboratory is
required to maintain performance records to define the quality of data that is generated.
8.1 .1 Before performing any analyses, the analyst must demonstrate the ability to generate
acceptable accuracy and precision with this method. This ability is established as
described in Section 8.2.
8.1.2 The laboratory must spike and analyze a minimum of 10% of all samples to monitor
continuing laboratory performance. This procedure is described in Section 8.4.
8.2 To establish the ability to generate acceptable accuracy and precision, the analyst must perform
the following operations.
8.2.1 Select a representative spike concentration for each compound to be measured.
8.2.2 Add the known amount of dithiocarbamate standard to each of a minimum of four
1000-mL aliquots of reagent water. A representative wastewater may be used in place of
the reagent water, but one or more additional aliquots must be analyzed to determine
background levels, and the spike level must exceed twice the background level for the
test to be valid. Analyze the aliquots according to the method beginning in Section 10.
8.2.3 Calculate the average percent recovery (R), and the standard deviation of the percent
recovery (s), for the results. Wastewater background corrections must be made before
R and s calculations are performed.
8.2.4 Using the appropriate data from Table 1, determine the recovery and single-operator
precision expected for the method, and compare these results to the values calculated in
Section 8.2.3. If the data are not comparable, review potential problem areas and repeat
the test.
8.3 The analyst must calculate method performance criteria and define the performance of the
laboratory for each spike concentration and parameter being measured.
8.3.1 Calculate upper and lower control limits for method performance as follows:
Upper Control Limit (UCL) = R + 3s
Lower Control Limit (LCL) = R - 3s
309
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Method 630
where R and s are calculated as in Section 8.2.3. The UCL and LCL can be used to
construct control charts 6 that are useful in observing trends in performance.
8.3.2 The laboratory must develop and maintain separate accuracy statements of laboratory
performance for wastewater samples. An accuracy statement for the method is defined
as R ± s. The accuracy statement should be developed by the analysis of four aliquots
of wastewater as described in Section 8.2.2, followed by the calculation of R and s.
Alternatively, the analyst may use four wastewater data points gathered through the
requirement for continuing quality control in Section 8.4. The accuracy statements
should be updated regularly. 6
8.4 The laboratory is required to collect in duplicate a portion of their samples to monitor spike
recoveries. The frequency of spiked sample analysis must be at least 10% of all samples or one
spiked sample per month, whichever is greater. One aliquot of the sample must be spiked and
analyzed as described in Section 8.2. ii the recovery for a particular parameter does not fall
within the control limits for method perfonnance, the results reported for that parameter in all
samples processed as part of the same set must be qualified as described in Section 11.3. The
laboratory should monitor the frequency of data so qualified to ensure that it remains at or
below 5%.
8.5 Before processing any samples, the analyst must demonstrate through the analysis of a 1-L aliquot
of reagent water that all glassware and reagent interferences are under control. Each time a set
of samples is extracted or there is a change in reagents, a laboratory reagent blank must be
processed as a safeguard against laboratory contamination.
8.6 It is recommended that the laboratory adopt additional quality assurance practices for use with this
method. The specific practices that are most productive depend upon the needs of the laboratory
and the nature of the samples. Field duplicates may be analyzed to monitor the precision of the
sampling technique. Whenever possible, the laboratory should perform analysis of quality control
materials and participate in relevant performance evaluation studies.
9. SAMPLE COLLECTION, PRESERVATiON, AND HANDLING
9.1 Grab samples must be collected in glass containers. Conventional sampling practices 7 should be
followed; however, the bottle must not be prerinsed with sample before collection. Composite
samples should be collected in refrigerated glass containers in accordance with the requirements
of the program. Automatic sampling equipment must be as free as possible of plastic and other
potential sources of contamination.
9.2 The samples must be iced or refrigerated at 4°C from the time of collection until extraction.
9.3 All samples must be analyzed within 7 days of collection.
10. SAMPLEANALYSIS
10.1 Assemble the hydrolysis apparatus as follows (see Figure 1):
10.1.1 Place the hydrolysis flask on the hot plate.
10.1.2 Place the addition funnel in one of the necks of the hydrolysis flask and the dust trap in
the top of the funnel.
310
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Method 630
10.1.3 Place the condenser in the other neck and attach two gas-washing bottles in succession
to the condenser outlet.
10.1.4 Attach a vacuum line with a flow valve to the second scrubber.
10.2 Allow the sample to warm to room temperature. Mark the water meniscus on the side of the
sample bottle for later determInation of sample volume. Pour the entire sample into the 2-L
hydrolysis flask. Rinse the bottle four times with 100-mL aliquots of reagent water, adding the
washes to the hydrolysis flask. Bring the volume in the hydrolysis flask to approximately
1,500 mL with reagent water.
10.3 Place 5.0 mL of color reagent into the CS 2 trap (second gas-washing bottle). Place 9 mL of zinc
acetate solution into the H 2 S scrubber (first gas washing bottle). Add 2 mL of ethanol to the H 2 S
scrubber. Place a magnetic stirring bar in the hydrolysis flask and place the flask on the
hotplate/magnetic stirrer (ambient at this time). Assemble the apparatus providing adequate
support for all glassware. The addition funnel stem opening must be below the water level.
Ground-glass joints may be slightly coated with silicone grease.
10.4 Start the stirrer, begin water flow through the condenser, and turn on hot plate and begin heating
the flask. Open the needle valve slightly and start the aspirator. By closing the needle valve,
adjust the airflow through the absorption train until the proper flow is attained. (The column of
bubbles extends to the bottom of the spherical expansion chamber at the top of the CS 2 trap.) Add
30 mL of decomposition reagent to the flask.
NO TE: The analyst must ensure that the sample pH is less than 2 during hydrolysis.
10.5 Bring the liquid in the flask to a gentle boil. Continue the boiling for 60 minutes, then remove
the heat. Continue aspiration until boiling ceases.
10.6 Transfer the contents of the CS 2 trap into a 25.0-mL volumetric flask by forcing the liquid through
the glass fit and out of the inlet arm with pressure from a large pipette bulb. Ensure quantitative
transfer by rinsing the trap three times with ethanol. Bring the colored solution to volume with
ethanol. Mix thoroughly and allow the color to develop for at least 15 minutes but not more than
2 hours before determining the absorbance.
10.7 Determine the absorbance of the sample at 435 nm and 380 nm using a 1-cm cell or a 4-cm cell
as necessary. Determine the weight of dithiocarbamate from the appropriate calibration curve
prepared in Section 7.4.
10.8 Determine the original sample volume by refilling the sample bottle to the mark and transferring
the liquid to a 1,000-mL graduated cylinder. Record the sample volume to the nearest 5 mL. If
a smaller measured aliquot of sample was used to remain within the range of the color reagent,
this step may be omitted.
311
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Method 630
11. CALCULA liONS
11.1 Determine the concentration of total dithiocarbamates in the sample as ziram directly from the
calibration curve. When a specific dithiocarbamate is being measured, quantitate in terms of the
selected pesticide.
11.2 Report results in micrograms per liter without correction for recovery data. When duplicate and
spiked samples are analyzed, report all data obtained with the sample results.
11.3 For samples processed as part of a set where the laboratory spiked sample recovery falls outside
of the control limits in Section 8.3, data for the affected parameters must be labeled as suspect.
12. METHOD PERFORMANCE
12.1 The method detection limit (MDL) is defined as the minimum concentration of a substance that
can be measured and reported with 99% confidence that the value is above zero. 1 The MDL
concentrations listed in Table I were determined using wastewater, and are expressed in
concentration units of the spiked materials.’
12.2 In a single laboratory, Environmental Science and Engineering, using spiked wastewater samples,
the average recoveries presented in Table 1 were obtained. The percent standard deviation of the
recovery is also included in Table 1.1 All recoveries are based on calibrations using the specific
dithiocarbamate instead of ziram..
312
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Method 630
References
1. “Pesticides Methods Development,” Report for EPA Contract 68-03-2897 (In preparation).
2. ASTM Annual Book of Standards, Part 31, D3694, “Standard Practice for Preparation of Sample
Containers and for Preservation, “ American Society for Testing and Materials, Philadelphia, PA,
p. 679, 1980.
3. “Carcinogens - Working with Carcinogens,” Department of Health, Education, and Welfare,
Public Health Service, Center for Disease Control, National Institute for Occupational Safety and
Health, Publication No. 77-206, Aug. 1977.
4. “OSHA Safety and Health Standards, General Industry,” (29 CFR 1910), Occupational Safety and
Health Administration, OSHA 2206, (Revised, January 1976).
5. “Safety in Academic Chemistry Laboratories,” American Chemical Society Publication,
Committee on Chemical Safety, 3rd Edition, 1979.
6. “Handbook for Analytical Quality Control in Water and Wastewater Laboratories,”
EPA-600/4-79-019, U. S. Environmental Protection Agency, Environmental Monitoring and
Support Laboratory - Cincinnati, Ohio 45268, March 1979.
7. ASTM Annual Book of Standards, Part 31, D3370, “Standard Practice for Sampling Water,”
American Society for Testing and Materials, Philadelphia, PA, p. 76, 1980.
8. Glaser, J .A. et.al, “Trace Analysis for Wastewaters,” Environmental Science & Technology, 15,
1426 (1981).
313
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Method 630
Table 1. Method Performance
Method
Detection Mean Standard
Limit Sample Number of Spike Recovery Deviation
Parameter (pg/L) Type* Replicates (pg/Li (%) (%i
Maneb 15.3 1 7 31.5 97.1 15.5
Methani 3.7 2 7 20.1 94.5 5.9
3 7 250.0 65.2 2.8
Ziram 1.9 4 8 32.2 100.0 2.0
5 8 1050.0 96.2 10.0
SampIe type:
I = Municipal wastewater
2 = Mixture of 13% industrial (pesticide manufacturing) wastewater and 87% municipal wastewater
3 = Industrial wastewater, pesticide manufacturing
4 = Mixture of 40% industrial and 60% municipal wastewater
5 = 7% industrial process water, 7% industrial wastewater, 86% municipal wastewater
314
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Method 630
Figure 1. Dithiocarbamate Hydrolysis Apparatus.
A52-002-80
315
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Method 604.1
16
-------
Method 630.1
The Determination of
Dithiocarbamates Pesticides in
Municipal and Industrial
Waste water
-------
Method 630.1
The Determination of Dithiocarbamates Pesticides in Municipal and
Industrial Waste water
SCOPE AND APPLICA TION
1.1 This method covers the determination of certain dithiocarbamates pesticides after conversion to
carbon disulfide. The following parameters can be determined by this method:
Parameter CAS. No.
Amobam 3566-10-7
Susan 40 51026-28-9
Busan 85 128-03-0
EXD 502-55-6
Ferbam 14484-64-1
KN Methyl 137-41-7
Metham 137-42-8
Nabam 142-59-6
Nabonate 138-93-2
Sodium dimethyldithiocarbamate 128-04-1
Thiram 137-26-8
Zineb 12122-67-7
Ziram 137-30-4
1.2 The compounds are decomposed to form carbon disulflde (CS 2 ) and the total dithiocarbamate
concentration is measured from the amount of CS 2 produced by acid hydrolysis. Unless the
sample can be otherwise characterized, all results are reported as ziram.
1.3 This is a total-residue gas chromatographic (GC) method applicable to the determination of the
compounds listed above in municipal and industrial discharges as provided under 40 CFR 136.1.
Any modification of this method beyond those expressly permitted shall be considered a major
modification subject to application and approval of alternative test procedures under 40 CFR 136.4
and 136.5.
1.4 The method detection limits (MDLs, defined in Section 14) for the parameters listed in Section 1.1
are listed in Table 1. The MDLs for a specific wastewater may differ from those listed, depending
upon the nature of interferences in the sample matrix.
1.5 This method is restricted to use by or under the supervision of analysts experienced in the use of
gas chromatography and in the interpretation of gas chromatograms. Each analyst must
demonstrate the ability to generate acceptable results with this method using the procedure
described in Section 8.2.
319
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Method 630.1
2. SUMMARY OF METHOD 1
2.1 A measured 5-mL volume of sample is digested with acid to yield CS 2 by hydrolysis of the
dithiocarbamate moiety. The evolved CS 2 is extracted from water into hexane. Gas
chromatographic conditions are described which permit the separation and measurement of CS 2
in the extract by gas chromatography with a Hall detector in the sulfur mode.
2.2 This method provides a cleanup procedure involving purging of any indigenous CS 2 from the
sample at pH 12 to 13. This procedure is performed using a vortex evaporator.
3.
IF,
3.1 Method interferences may be caused by contaminants in solvents, reagents, glassware, and other
sample-processing apparatus that lead to discrete artifacts or elevated baselines in gas
chromatograms. All reagents and apparatus must be routinely demonstrated to be free from
interferences under the conditions of the analysis by running laboratory reagent blanks as
described in Section 8.5.
3.1.1
Glassware must be scrupulously cleaned. 2 Clean all glassware as soon as possible after
use by thoroughly rinsing with the last solvent used in it. Follow by washing with hot
water, drain dry, and heat in an oven or muffle furnace at 4.00°C for 15 to 30 minutes.
Do not heat volumetric ware. Some thermally stable materials, such as PCBs, may not
be eliminated by this treatment. Thorough rinsing with acetone and pesticide-quality
hexane may be substituted for the heating. After drying and cooling, seal and store
glassware in a clean environment to prevent any accumulation of dust or other
contaminants. Store inverted or capped with aluminum foil.
3.1.2 The use of high-purity reagents and solvents helps to minimize interference problems.
Purification of solvents by distillation in all-glass systems may be required.
3.2 Carbon disulfide may be a direct interferent in wastewaters. This method includes procedures to
purge CS 2 from the wastewater prior to acid hydrolysis of the sample. A vortex evaporator is
used for CS 2 removal.
3.3 Additional matrix interferences may be caused by contaminants that are coextracted from the
sample and from other CS 2 generating compounds. The extent of matrix interferences will vary
considerably from source to source, depending upon the nature of the sample.
4. SAFETY
4.1 The toxicity or carcinogenicity of each reagent used in this method has not been precisely defined;
however, each chemical compound should be treated as a potential health hazard. From this
viewpoint, exposure to these chemicals must be reduced to the lowest possible level by whatever
means available. The laboratory is responsible for maintaining a current awareness file of OSHA
regulations regarding the safe handling of the chemicals specified in this method. A reference file
of material data handling sheets should also be made available to all personnel involved in the
chemical analysis. Additional references to laboratory safety are available and have been
identified” for the information of the analyst.
4.2 Nabam (ethylene bis (dithiocarbamate)) has been identified as having substantial evidence of
carcinogenicity and should be handled according to OSHA regulations.
320
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Method 630.1
5. APPARA TI /S AND MA TERIALS
5.1 Sampling equipment, for discrete or composite sampling.
5.1 .1 Sample containers: 40-mL screw-cap vials (Pierce No. 13075 or equivalent): each
equipped with a polytetrafluoroethylene (PTFE)-faced silicone septum (Pierce No. 12722
or equivalent). Prior to use, wash vials and septa with detergent and rinse with tap and
distilled water. Allow the vials and septa to air dry at room temperature, place in a
105°C oven for 1 hour, then remove and allow to cool in an area known to be free of
organics.
5.1.2 Automatic sampler (optional): Must incorporate glass sample containers for the
collection of a minimum of 250 mL. Sample containers must be kept refrigerated at 4°C
and protected from light during compositing. If the sampler uses a peristaltic pump, a
minimum length of compressible silicone rubber tubing may be used. Before use,
however, the compressible tubing should be thoroughly rinsed with methanol, followed
by repeated rinsings with distilled water to minimize the potential fOr contamination of
the sample. An integrating flow meter is required to collect flow-proportional
composites.
5.2 Glassware.
5.2.1 Centrifuge tube: 15-mL, conical, with PTFE-lined screw-cap.
5.2.2 Volumetric flask: 250-mL with glass stopper.
5.2.3 Bottles: 100- to 200-mL capacity with PTFE-lined screw-caps.
5.3 Vortex Evaporator: Buchler 3-2200, equipped with sample block to hold 36 15-mL conical-
bottom centrifuge tubes and appropriate vacuum cover.
5.4 Water bath: Heated, capable of temperature control (±2°C). The bath should be used in a hood.
5.5 Balance: Analytical, capable of accurately weighing to the nearest 0.0001 g.
5.6 Gas chromatograph: Analytical system complete with gas chromatograph suitable for on-column
injection and all required accessories including syringes, analytical columns, gases, detector, and
strip-chart recorder. A data system is recommended for measuring peak areas.
5.6.1 Column: 180 cm by 2 mm ID glass, packed with 0.1 % SP-1000 on Carbopack C
(80/ 100 mesh) or equivalent. This column was used to develop the method performance
statements in Section 14. Alternative columns may be used in accordance with the
provisions described in Section 11.1.
5.6.2 Detector: Hall detector operated in the sulfur mode. This detector has proven effective
in the analysis of wastewaters for the compounds listed in the scope and was used to
develop the method performance statements in Section 14.
6. REAGENTS
6.1 Reagent water: Reagent water is defined as a water in which an interferent is not observed at the
MDL of each parameter of interest.
6.2 Hexane: Distilled-in-glass quality or equivalent.
6.3 Sulfuric acid, 12N: Slowly add 100 mL concentrated sulfuric acid to 200 mL reagent water.
321
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Method 630.1
6.4 Sodium phosphate, tribasic, dodeca-hydrate: Baker reagent grade or equivalent.
6.5 Tribasic sodium phosphate, 0. 1M: Dissolve 38 g of tribasic sodium phosphate in reagent water
and dilute to 1000 mL with reagent water.
6.6 Stannous chloride: SnC I 2 2H 2 O, ACS grade.
6.7 Stannous chloride reagent: Dissolve 1.5 g stannous chloride in 100 mL 12N sulfuric acid.
Prepare fresh daily.
6.8 Sodium chloride: Heated at 45°C for 8 hours.
6.9 Stock standard solutions (0.1 p gI L): Stock standard solutions can be prepared from pure
standard materials or purchased as certified solutions.
6.9.1 Prepare dithiocarbamate spiking solutions by accurately weighing about 0.025 g of pure
material. Dissolve the material in 0. 1M Na 3 PO 4 and dilute to volume in a 250-mL
volumetric flask. Larger volumes can be used at the convenience of the analyst. If
compound purity is certified at 96% or greater, the weight can be used without correction
to calculate the concentration of the stock standard. Commercially prepared stock
standards can be used at any concentration if they are certified by the manufacturer or
by an independent source.
6.9.2 (0.1 &g/ tL) Prepare CS 2 stock standard solution by adding 7.9 L of CS 2 to hexane and
diluting to volume in a lOO-mL volumetric flask.
6.9.3 Transfer the stock standard solutions into PTFE-sealed screw-cap bottles. Store at 4°C.
Frequently check standard solutions for signs of degradation or evaporation.
6.9.4 Stock standard solutions must be replaced after 6 months, or sooner if comparison with
check standards indicates a problem.
7. CAU8RA TION
7.1 Use ziram as the standard for total dithiocarbamates when a mixture of dithiocarbamates is likely
to be present. Use the specific dithiocarbamate as a standard when only one pesticide is present
and its identity has been established.
7.2 Establish gas chromatographic operating parameters equivalent to those indicated in Table 1. The
gas chromatographic system can be calibrated using the external standard technique (Section 7.3).
7.3 External standard calibration procedure:
7.3.1 Prepare calibration standards at a minimum of three concentration levels by adding
volumes of the CS2 stock standard to a volumetric flask and diluting to volume with
hexane. One of the external standards should be at a concentration near, but above, the
method detection limit. The other concentrations should correspond to the range of
concentrations expected in the sample concentrates or should define the working range
of the detector.
7.3.2 Using injections of 1 to 5 &L of each calibration standard, tabulate peak height or area
responses against the mass injected. The results can be used to prepare a calibration
curve for CS 2 . Alternatively, the ratio of the response to the mass injected, defined as
the calibration factor (CF), can be calculated at each standard concentration. If the
322
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Method 630.1
relative standard deviation of the calibration factor is less than 10% over the working
range, the average calibration factor can be used in place of a calibration curve.
7.3.3 The working calibration curve or calibration factor must be verified on each working
shift by the measurement of one or more calibration standards. If the response for CS 2
varies from the predicted response by more than ± 10%, the test must be repeated using
a fresh calibration standard. Alternatively, a new calibration curve or calibration factor
must be prepared.
7.4 Before using any cleanup procedure, the analyst must process a series of calibration standards
through the procedure to validate the absence of interferences from the reagents.
8. QuAurY CONTROL
8.1 Each laboratory using this method is required to operate a formal quality control program. The
minimum requirements of this program consist of an initial demonstration of laboratory capability
and the analysis of spiked samples as a continuing check on performance. The laboratory is
required to maintain performance records to define the quality of data that is generated.
8.1.1 Before performing any analyses, the analyst must demonstrate the ability to generate
acceptable accuracy and precision with this method. This ability is established as
described in Section 8.2.
8.1.2 In recognition of the rapid advances occurring in chromatography, the analyst is
permitted certain options to improve the separations or lower the cost of measurements.
Each time such modifications to the method are made, the analyst is required to repeat
the procedure in Section 8.2.
8.1.3 The laboratory must spike and analyze a minimum of 10% of all samples to monitor
continuing laboratory performance. This procedure is described in Section 8.4.
8.2 To establish the ability to generate acceptable accuracy and precision, the analyst must perform
the following operations.
8.2.1 Select a representative spike concentration for each compound to be measured.
8.2.2 Add a known amount of an individual dithiocarbamate standard to a minimum of four
5-niL aliquots of 0. 1M tribasic sodium phosphate. A representative wastewater may be
used in place of the reagent water, but one or more additional aliquots must be analyzed
to determine background levels, and the spike level must exceed twice the background
level for the test to be valid. Analyze the aliquots according to the method beginning in
Section 10.
8.2.3 Calculate the average percent recovery (R), and the standard deviation of the percent
recovery (s), for the results. Wastewater background corrections must be made before
R and s calculations are performed.
8.2.4 Using the appropriate data from Table 2, determine the recovery and single-operator
precision expected for the method, and compare these results to the values measured in
Section 8.2.3. If the data are not comparable, the analyst must review potential problem
areas and repeat the test.
8.3 The analyst must calculate method performance criteria and define the performance of the
laboratory for each spike concentration and parameter being measured.
323
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Method 630.1
8.3.1 Calculate upper and lower control limits for method performance as follows:
Upper Control Limit (UCL) = R + 3s
Lower Control Limit (LCL) = R - 3s
where R and s are calculated as in Section 8.2.3. The UCL and LCL can be used to
construct control charts 6 that are useful in observing trends in performance.
8.3.2 The laboratory must develop and maintain separate accuracy statements of laboratory
performance for wastewater samples. An accuracy statement for the method is defined
as R ± s. The accuracy statement should be developed by the analysis of four aliquots
of wastewater as described in Section 8.2.2, followed by the calculation of R and s.
Alternatively, the analyst may use four wastewater data points gathered through the
requirement for continuing quality control in Section 8.4. The accuracy statements.
should be updated regularly. 6
8.4 The laboratory is required to collect in duplicate a portion of their samples to monitor spike
recoveries. The frequency of spiked sample analysis must be at least 10% of all samples or one
sample per month, whichever is greater. One aliquot of the sample must be spiked and analyzed
as described in Section 8.2. If the recovery for a particular compound does not fall within the
control limits for method performance, the results reported for that compound in all samples
processed as part of the same set must be qualified as described in Section 12.3. The laboratory
should monitor the frequency of data so qualified to ensure that it remains at or below 5%.
8.5 Before processing any samples, the analyst should demonstrate through the analysis of a 5-mL
aliquot of 0. 1M tribasic sodium phosphate that all glassware and reagent interferences are under
control. Each time a set of samples is extracted or there is a change in reagents, a laboratory
reagent blank should be processes as a safeguard against laboratory contamination.
8.6 It is recommended that the laboratory adopt additional quality assurance practices for use with this
method. The specific practices that are most productive depend upon the needs of the laboratory
and the nature of the samples. Field duplicates may be analyzed to monitor the precision of the
sampling technique. When doubt exists over the identification of a peak on the chromatogram,
confirmatory techniques such as gas chromatography with a dissimilar column, specific element
detector, or mass spectrometer must be used. Whenever possible, the laboratory should perform
analysis of standard reference materials and participate in relevant performance evaluation studies.
9. SAMPLES COLLECTiON, PRESERVATION, AND HANDLING
9.1 Grab samples must be collected in glass containers. Conventional sampling practices 7 should be
followed; however, the bottle must not be prerinsed with sample before collection. Composite
samples should be collected in refrigerated glass containers in accordance with the requirements
of the program. Automatic sampling equipment must be as free as possible of plastic and other
potential sources of contamination.
9.2 The samples must be iced or refrigerated at 4°C from the time of collection until extraction.
9.3 Add 15.2 g of tribasic sodium phosphate per 40 mL of sample to the sample to adjust pH to 12
to 13 at time of collection.
324
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Method 630.1
10. SAMPLE CLEANUP AND EXTRACTION
10.1 Place 5 mL of sample in a 15-mL conical centrifuge tube.
10.2 Add 0.75 g of NaC1 and shake tube to dissolve salt.
10.3 Add 2 mL of MTBE and process in a vortex evaporator for 10 minutes with the temperature at
30°C, a vacuum of 30 inches Hg, and the vortex speed control set at 4.5.
10.4 Repeat step in Section 10.3 twice.
10.5 Add 0.75 mL of hexane and 2.5 mL of SnC1 2 reagent to the aqueous layer. Cap tube tightly and
invert in a water bath at 50°C for 30 minutes.
10.6 Remove tube from water bath and let cool inverted to room temperature.
10.7 Shake tube for 1 minute without venting. Analyze the hexane layer by GC with a Hall detector
in the sulfur mode. If CS 2 levels are outside of the GC calibration range, the sample can be
diluted a known amount with hexane and reanalyzed.
11. GAS CHROMATOGRAPHY
11.1 Table I summarizes the recommended operating conditions for the gas chromatograph. Included
in this table are estimated retention time and MDLs that can be achieved by this method. An
example of the chromatography achieved from Column 1 is shown in Figure 1. Other packed
columns, chromatographic conditions, or detectors may be used if the requirements of Section 8.2
are met. Capillary (open-tubular) columns may also be used if the relative standard deviations
of responses for replicate injections are demonstrated to be less than 6% and the requirements of
Section 8.2 are met.
1 1.2 Calibrate the gas chromatographic system daily as described in Section 7.
1 1.3 Inject 1 to 5 gzL of the sample extract using the solvent flush technique. 8 Record the volume
injected to the nearest 0.05 1 iL, and the resulting peak sizes in area or peak height units. An
automated system that consistently injects a constant volume of extract may also be used.
11.4 The width of the retention-time window used to make identifications should be based upon
measurements of actual retention-time variations of standards over the course of a day. Three
times the standard deviation of a retention time for a compound can be used to calculate a
suggested window size; however, the experience of the analyst should weigh heavily in the
interpretation of chromatograms.
1 1.5 If the response for the peak exceeds the working range of the system, dilute the extract with
hexane and reanalyze.
11.6 If the measurement of the peak response is prevented by the presence of interferences, further
cleanup is required.
12. CALCULATIONS
12.1 Determine the concentration of carbon disulfide in the sample.
12.1.1 If the external standard calibration procedure is used, calculate the amount of material
injected from the peak response using the calibration curve or calibration factor in
325
-------
Method 630.1
Section 7.2.2. The concentration of dithiocarbainate in the sample can be calculated as
follows:
:
Equation 1
(A)(V,)(M )
Concentration, zg/L =
where
A = Aniowit of CS 2 injected, in ng
V 1 = Volume of extract injected, in ig/L
= Volume of total extract, in pL
V 1 = Volume of ter extracted, in niL
U = Molecular weight of dUhiocarbaniate
C = Theoretical number of moles of C S 2 jbnned per mole of dithicoabamate
12.2 Determine the concentration of total dithiocarbamates in the sample as ziram. When a specific
dithiocarbamate is being measured, quantitate in terms of the selected pesticide.
12.3 Report results in micrograms per liter without correction for recovery data. When duplicate and
spiked samples are analyzed, report all data obtained with the sample results.
12.4 For samples processed as part of a set where the laboratory spiked sample recovery falls outside
of the control limits in Section 8.3, data for the affected compounds must be labeled as suspect.
13. METHOD PERFORMANCE
13.1 The MDL is defined as the minimum concentration of a substance that can be measured and
reported with 99% confidence that the value is above zero. 9 The MDL concentrations listed in
Table 1 were obtained using spiked reagent water samples. 1
13.2 This method has been tested for linearity of recovery from spiked reagent water and has been
demonstrated to be applicable over the concentration range from 10 gIL to 1000 g/L.
13.3 In a single laboratory, Battelle Columbus Laboratories, using spiked wastewater samples, the
average recoveries of the parameters listed in Section 1.1 presented in Table 2 were obtained.
Seven replicates of the wastewater were spiked and analyzed. The standard deviation of the
percent recovery is also included in Table 2.’
326
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Method 630.1
References
1. “Determination of Pesticides and Priority Pollutants in Industrial and Municipal Wastewaters,”
EPA Contract Report 68-03-1760, Work Assignment 4 (in preparation).
2. ASTM Annual Book of Standards, Part 31, D3694, “Standard Practice for Preparation of Sample
Containers and for Preservation,” American Society for Testing and Materials, Philadelphia,
Pennsylvania, p. 679, 1980.
3. “Carcinogens - Working with Carcinogens,” Department of Health, Education, and Welfare,
Public Health Service, Center for Disease Control, National Institute for Occupational Safety and
Health, Publication No. 77-206, August, 1977..
4. “OSHA Safety and Health Standards, General Industry,” (29 CFR 1910), Occupational Safety and
Health Administration, OSHA 2206 (Revised, January 1976).
5. “Safety in Academic Chemistry Laboratories,” American Chemical Society Publication,
Committee on Chemical Safety, 3rd Edition, 1979.
6. “Handbook for Analytical Quality Control in Water and Wastewater Laboratories,”
EPA-60014-79-019, U.S. Environmental Protection Agency, Environmental Monitoring and
Support Laboratory Cincinnati, Ohio, March 1979.
7. ASTM Annual Book of Standards, Part 31, D3370, “Standard Practice for Sampling Water,”
American Society for Testing and Materials, Philadelphia, Pennsylvania, p. 76, 1980.
8. Burke, J.A., “Gas Chromatography for Pesticide Residue Analysis; Some Practical Aspects,”
Journal of the Association of Official Analytical Chemists, 48, 1037 (1965).
9. Glaser, J.A. et al, “Trace Analysis for Wastewaters”, Environmental Science and Technology, 15,
1426 (1981).
327
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Method 630.1
Table 1. Chromatographic Conditions and Method Detection Limits
Parameter Retention Time MDL
(min.)(a) (pg/Li
Amobam 1.3 1.1
Busan4O 1.3 4.4
Busan85 1.3 1.3
EXD 1.3 5.2
Ferbam 1.3 2.9
KNMethyl 1.3 2.7
Metham 1.3 3.1
Nabam 1.3 1.6
Nabonate 1.3 0.9
Na DMDTC 1.3 2.8
Thiram 1.3 2.2
Zineb 1.3 4.1
Ziram 1.3 4.6
(a) Retention time of CS 2 under the following conditions: Carbopack C (80/100 mesh) coated with 0.1 %
Sp-1000 packed in a 180 cm long by 2 mm II) glass column with helium carrier gas at a flow rate of
25 mL/min. Column temperature held at 7°C for 3 minutes, programmed at 20°C/mm to 120°C, and
then held at 120°C for 5 minutes. Column effluent is vented from the Hall detector after elution of CS 2
from the column. Injector temperature and detector temperatures are 200°C. The Hall detector is
operated in the sulfur mode following manufacturer’s specifications.
328
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Method 630.1
Table 2. Single-Laboratory Accuracy and Precision
Parameter Sample Background Spike Mean Standard Number of
Type(a) (pg/LI (pg/LI Recovery Deviation Replicates
1%)
Amobam 1 4.6 50 90 7.8 7
1 4.6 500 93 8.7 7
Busan40 1 6.6 50 110 7.2 7
1 6.6 500 100 6.1 7
Busan 85 1 5.9 50 110 5.5 7
1 5.9 500 100 2.0 7
EXE) 1 4.5 50 71 7.5 7
1 4.5 500 76 2.4 7
Ferbam 1 5.2 50 94 4.8 7
1 5.2 500 110 1.8 7
KN Methyl 1 5.4 50 90 6.1 7
1 5.4 500 89 2.5 7
Methan 1 6.2 50 110 5.2 7
1 6.2 500 84 5.9 7
Nabam 1 4.8 50 62 6.6 7
1 4.8 500 65 13 7
Nabonate 1 6.1 50 66 11 7
1 6.1 500 56 12 7
Na DMDTC 1 5.4 50 110 2.5 7
1 5.4 500 110 4.2 7
Thiram 1 4.5 50 89 2.9 7
1 4.5 500 82 3.4 7
Zineb 1 5.2 50 87 3.4 7
1 5.2 500 86 9.4 7
Ziram 1 5.7 50 100 12 7
1 5.7 500 95 19 7
(a) 1 = Wastewater from a manufacturer of a dithiocarbamate diluted 1000:1 with Columbus POTW
secondary effluent.
329
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Method . I
Figure 1. GC-HALL Chromatogram of 0.1 ng of CS 2 .
CS 2
I’ ‘
1.1 1.2
I I I I I I I
1.3 1.4 1.5 1.6
Retention Time (minutes)
I I I I
1.7 1.8 1.9
I I
2.0
M2 41
330
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Method 631
The Determination of Benom yl
and Carbendazim in Municipal
and Industrial Wastewater
-------
Method 631
The Determination of Benomyl and Carbendazim in Municipal and
Industrial Wastewater
1. SCOPE AND APPL1CA TION
1.1 This method covers the determination of benomyl and carbendazim. The following parameters
can be determined by this method:
Parameter Storet No. CAS No.
Benomyl — 17804-35-2
Carbendazim — 10605-21-7
1.2 Benomyl cannot be determined directly by this method. Benomyl is hydrolyzed to carbendazim,
and both compounds are measured and reported as carbendazim.
1.3 This is a high performance liquid chromatographic (HPLC) method applicable to the determination
of the compounds listed above in industrial and municipal discharges as provided under 40 CFR
136.1. Any moditication of this method beyond those expressly permitted shall be considered a
major modification subject to application and approval of alternate test procedures under
40 CFR 136.4 and 136.5.
1.4 The method detection limit (MDL, defined in Section 15) for each parameter is 8.7 p g/L. The
MDL for a specific wastewater may differ from those listed, depending upon the nature of inter-
ferences in the sample matrix.
1.5 This method is restricted to use by or under the supervision of analysts experienced in the use of
liquid chromatography and in the interpretation of liquid chromatograms. Each analyst must
demonstrate the ability to generate acceptable results with this method using the procedure
described in Section 8.2.
1.6 When this method is used to analyze unfamiliar samples for either of the compounds above,
compound identifications should be supported by at least one additional qualitative technique.
2. SUMMARY OF METHOD
2.1 A measured volume of sample, approximately 1 L, is acidified if necessary to hydrolyze benomyl
to carbendazim. The total carbendazim is extracted with methylene chloride using a separatory
funnel. The extract is dried and exchanged to methanol during concentration to a volume of 10
mL or less. HPLC conditions are described which permit the separation and measurement of total
carbendazim in the extract by HPLC with a UV detector.” 2
3. INTERFERENCES
3.1 Method interferences may be caused by contaminants in solvents, reagents, glassware, and other
sample-processing apparatus that lead to discrete artifacts or elevated baselines in liquid chromato-
grams. All reagents and apparatus must be routinely demonstrated to be free from interferences
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Method 631
under the conditions of the analysis by running laboratory reagent blanks as described in
Section 85.
3.1.1 Glassware must be scrupulously cleaned. 3 Clean all glassware as soon as possible after
use by thoroughly rinsing with the last solvent used in it. Follow by washing with hot
water and detergent and thorough rinsing with tap and reagent water. Drain dry, and heat
in an oven or muffle furnace at 400°C for 15 to 30 minutes. Do not heat volumetric
ware. Thermally stable materials, such as PCBs, may not be eliminated by this
treatment. Thorough rinsing with acetone and pesticide-quality hexane may be substituted
for the heating. After drying and cooling, seal and store glassware in a clean
environment to prevent any accumulation of dust or other contaminants. Store inverted
or capped with aluminum foil.
3.1.2 The use of high-purity reagents and solvents helps to minimize interference problems.
Purification of solvents by distillation in all-glass systems may be required.
3.2 Matrix interferences may be caused by contaminants that are coextracted from the sample. The
extent of matrix interferences will vary considerably from source to source, depending upon the
nature and diversity of the industrial complex or municipality sampled. Unique samples may
require cleanup approaches to achieve the MDL listed in Section 1.
4. SAFETY
4.1 The toxicity or carcinogenicity of each reagent used in this method has not been precisely defined;
however, each chemical compound must be treated as a potential health hazard. From this
viewpoint, exposure to these chemicals must be reduced to the lowest possible level by whatever
means available. The laboratory is responsible for maintaining a current awareness file of OSHA
regulations regarding the safe handling of the chemicals specified in this method. A reference file
of material data handling sheets should also be made available to all personnel involved in the
chemical analysis. Additional references to laboratory safety are available and have been
identified for the information of the analyst.
5. APPARA TUS AND MA TER/ALS
5.1 Sampling equipment, for discrete or composite sampling.
5.1.1 Grab-sample bottle: Amber borosilicate or flint glass, 1-L or 1-quart volume, fitted with
screw-caps lined with TFE-fluorocarbon. Aluminum foil may be substituted for TFE if
the sample is not corrosive. If amber bottles are not available, protect samples from
light. The container and cap liner must be washed, rinsed with acetone or methylene
chloride, and dried before use to minimize contamination.
5.1.2 Automatic sampler (optional): Must incorporate glass sample containers for the
collection of a minimum of 250 mL. Sample containers must be kept refrigerated at 4°C
and protected from light during compositing If the sampler uses a peristaltic pump, a
minimum length of compressible silicone rubber tubing may be used. Before use,
however, the compressible tubing must be thoroughly rinsed with methanol, followed by
repeated rinsings with reagent water to minimize the potential for contamination of the
sample. An integrating flow meter is required to collect flow-proportional composites.
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Method 631
5.2 Glassware. (All specifications are suggested. Catalog numbers are included for illustration only.)
5.2.1 Separatory funnel: 250-mL, with TFE-fluorocarbon stopcock, ground-glass or TFE
stopper.
5.2.2 Drying column: Chromatographic column 4.00 mm long by 19 mm ID with coarse-fritted
disc.
5.2.3 Concentrator tube, Kuderna-Danish: 10-mL, graduated (Kontes K-570050-1025 or
equivalent). Calibration must be checked at the volumes employed in the test. Ground-
glass stopper is used to prevent evaporation of extracts.
5.2.4 Evaporative flask, Kuderna-Danish: 500-mL (Kontes K-570001-0500 or equivalent).
Attach to concentrator tube with springs.
5.2.5 Snyder column, Kuderna-Danish: Three-ball macro (Kontes K-503000-0121 or
equivalent).
5.2.6 Vials: Amber glass, 10- to 15-mL capacity with TFE-fluorocarbon-Iined screw-cap.
5.3 Boiling chips: Approximately 10/40 mesh. Heat at 400°C for 30 minutes or perform a Soxhlet
extraction with methylene chloride.
5.4 Water bath: Heated, with concentric ring cover, capable of temperature control (±2°C). The
bath should be used in a hood.
5.5 Balance: Analytical, capable of accurately weighing to the nearest 0.0001 g.
5.6 Liquid chromatograph: High performance analytical system complete with high pressure syringes
or sample injection loop, analytical columns, detector and strip-chart recorder. A guard column
is recommended for all applications.
5.6.1 Column: 30 cm long by 4 mm ID stainless steel, packed with & Bondapak C 18 (10 ) or
equivalent. This column was used to develop the method performance statements in
Section 14. Alternative columns may be used in accordance with the provisions
described in Section 12.1.
5.6.2 Detector: Ultraviolet, 254 nm. This detector has proven effective in the analysis of
wastewaters and was used to develop the method performance statements in Section 14.
Alternative detectors may be used in accordance with the provisions described in
Section 12.1.
6. REAGENTS
6.1 Reagent water: Reagent water is defined as a water in which an interferent is not observed at the
method detection limit of each parameter of interest.
6.2 Methylene chloride, methanol: Pesticide-quality or equivalent.
6.3 Sodium sulfate: ACS, granular, anhydrous. Condition by heating in a shallow tray at 400°C for
a minimum of 4 hours to remove phthalates and other interfering organic substances.
Alternatively, heat 16 hours at 450 to 500°C in a shallow tray or perform a Soxhlet extraction
with methylene chloride for 48 hours.
6.4 Sodium hydroxide solution (iON): Dissolve 40g NaOH in reagent water and dilute to 100 mL.
6.5 Sulfuric acid solution (1+1): Slowly add 50 mL H 2 S0 4 (sp. gr. 1.84) to 50 mL of reagent water.
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Method 631
6.6 Mobile phase: Methanol/water (1+1). Mix equal volumes of HPLCIUV quality methanol and
reagent water.
6.7 Stock standard solution (1.00 ,Lg4LL): The stock standard solution may be prepared from a pure
standard material or purchased as a certified solution.
6.7.1 Prepare the stock standard solution by accurately weighing approximately 0.0100 g of
pure carbendazim. Dissolve the material in HPLCIUV quality methanol and dilute to
volume in a 10-mL volumetric flask. Larger volumes may be used at the convenience
of the analyst. If compound purity is certified at 96% or greater, the weight may be used
without correction to calculate the concentration of the stock standard. Commercially
prepared stock standards may be used at any concentration if they are certified by the
manufacturer or by an independent source.
6.7.2 Transfer the stock standard solution into a TFE-fluorocarbon-sealed screw-cap vial.
Store at 4°C and protect from light. Frequently check stock standard solutions for signs
of degradation or evaporation, especially just prior to preparing calibration standards
from them.
6.7.3 The stock standard solution must be replaced after 6 months, or sooner if comparison
with a check standard indicates a problem.
7. CALIBRATiON
7.1 Establish HPLC operating parameters equivalent to those indicated in Table 1. The HPLC system
may be calibrated using either the external standard technique (Section 7.2) or the internal
standard technique (Section 7.3).
7.2 External standard calibration procedure.
7.2.1 Prepare calibration standards at a minimum of three concentration levels by adding
accurately measured volumes of carbendazim stock standard to volumetric flasks and
diluting to volume with methanol. One of the external standards should be representative
of a concentration near, but above, the method detection limit. The other concentrations
should correspond to the range of concentrations expected in the sample concentrates or
should define the working range of the detector.
7.2.2 Using injections of 10 iL of each calibration standard, tabulate peak height or area
responses against the mass injected. The results can be used to prepare a calibration
curve for carbendazim. Alternatively, the ratio of the response to the mass injected,
defined as the calibration factor (CF), may be calculated for carbendazim at each standard
concentration. If the relative standard deviation of the calibration factor is less than 10%
over the working range, the average calibration factor can be used in place of a
calibration curve.
7.2.3 The working calibration curve or calibration factor must be verified on each working
shift by the measurement of one or more calibration standards. If the response for any
parameter varies from the predicted response by more than ±10%, the test must be
repeated using a fresh calibration standard. Alternatively, a new calibration curve or
calibration factor must be prepared.
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Method 631
7.3 Internal standard calibration procedure: To use this approach, the analyst must select an internal
standard similar to carbendazim in analytical behavior. The analyst must further demonstrate that
the measurement of the internal standard is not affected by method or matrix interferences. Due
to these limitations, no internal standard applicable to all samples can be suggested.
7.3.1 Prepare calibration standards at a minimum of three concentration levels of carbendazim
by adding volumes of stock standard to volumetric flasks. To each calibration standard,
add a known constant amount of internal standard, and dilute to volume with methanol.
One of the standards should be representative of a concentration near, but above, the
method detection limit. The other concentrations should correspond to the range of
concentrations expected in the sample concentrates, or should define the working range
of the detector.
7.3.2 Using injections of 10 j L of each calibration standard, tabulate the peak height or area
responses against the concentration for each compound and internal standard. Calculate
response factors (RF) for each compound as follows:
Equation 1
RF = ( A 5 )(C, )
(Á)(ç)
where
A 5 = Response for the parameter to be measured
A Response for the internal standard
C = Concentration of the internal standard, in 1 ig/L
= Concentration of the parameter to be measured, in g g/L
If the RF value over the working range is constant, less than 10% relative standard
deviation, the RF can be assumed to be invariant and the average RF may be used for
calculations. Alternatively, the results may be used to plot a calibration curve of
response ratios, A /A 1 against RF.
7.3.3 The working calibration curve or RF must be verified on each working shift by the
measurement of one or more calibration standards. If the response for carbendazim
varies from the predicted response by more than ± 10%, the test must be repeated using
a fresh calibration standard. Alternatively, a new calibration curve must be prepared.
7.4 Before using any cleanup procedure, the analyst must process a series of calibration standards
through the procedure to validate elution patterns and the absence of interference from the
reagents.
8. QuALITY CONTROL
8.1 Each laboratory using this method is required to operate a formal quality control program. The
minimum requirements of this program consist of an initial demonstration of laboratory capability
and the analysis of spiked samples as a continuing check on performance. The laboratory is
required to maintain performance records to define the quality of data that is generated.
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Method 631
8.1.1 Before performing any analyses, the analyst must demonstrate the ability to generate
acceptable accuracy and precision with this method. This ability is established as
described in Section 8.2.
8.1.2 In recognition of the rapid advances occurring in chromatography, the analyst is
permitted certain options to improve the separations or lower the cost of measurements.
Each time such modifications to the method are made, the analyst is required to repeat
the procedure in Section 8.2.
8.1.3 The laboratory must spike and analyze a minimum of 10% of all samples to monitor
continuing laboratory performance. This procedure is described in Section 8.4.
8.2 To establish the ability to generate acceptable accuracy and precision, the analyst must perform
the following operations.
8.2.1 Select a representative spike concentration for each compound to be measured. Using
stock standards, prepare a quality control check sample concentrate of either benomyl or
carbendazim in methanol, 1000 times more concentrated than the selected concentrations.
8.2.2 Using a pipette, add 1.00 mL of the check sample concentrate to each of a minimum of
four 1000-mL aliquots of reagent water. A representative wastewater may be used in
place of the reagent water, but one or more additional aliquots must be analyzed to
determine background levels, and the spike level must exceed twice the background level
for the test to be valid. Analyze the aliquots according to the method beginning in
Section 10.
8.2.3 Calculate the average percent recovery (R), and the standard deviation of the percent
recovery (s), for the results. Wastewater background corrections must be made before
R and s calculations are performed.
8.2.4 Using the appropriate data from Table 2, determine the recovery and single-operator
precision expected for the method, and compare these results to the values calculated in
Section 8.2.3. If the data are not comparable, review potential problem areas and repeat
the test.
8.3 The analyst must calculate method performance criteria and define the performance of the
laboratory for each spike concentration and parameter being measured.
8.3.1 Calculate upper and lower control limits for method performance as follows:
Upper Control LImit (UCL) = R + 3s
Lower Control Limit (LCL) = R - 3s
where R and s are calculated as in Section 8.2.3. The UCL and LCL can be used to
construct control charts 7 that are useful in observing trends in performance.
8.3.2 The laboratory must develop and maintain separate accuracy statements of laboratory
performance for wastewater samples. An accuracy statement for the method is defined
as R ± s. The accuracy statement should be developed by the analysis of four aliquots
of wastewater as described in Section 8.2.2, followed by the calculation of R and s.
Alternatively, the analyst may use four wastewater data points gathered through the
requirement for continuing quality control in Section 8.4. The accuracy statements
should be updated regularly. 7
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Method 631
8.4 The laboratory is required to collect in duplicate a portion of their samples to monitor spike
recoveries. The frequency of spiked sample analysis must be at least 10% of all samples or one
spiked sample per month, whichever is greater. One aliquot of the sample must be spiked and
analyzed as described in Section 8.2. If the recovery for benomyl or carbendazim does not fall
within the control limits for method performance, the results reported for that parameter in all
samples processed as part of the same set must be qualified as described in Section 13.3. The
laboratory should monitor the frequency of data so qualified to ensure that it remains at or
below 5%.
8.5 Before processing any samples, the analyst must demonstrate through the analysis of a 1-L aliquot
of reagent water that all glassware and reagent interferences are under control. Each time a set
of samples is extracted or there is a change in reagents, a laboratory reagent blank must be
processed as a safeguard against laboratory contamination.
8.6 It is recommended that the laboratory adopt additional quality assurance practices for use with this
method. The specific practices that are most productive depend upon the needs of the laboratory
and the nature of the samples. Field duplicates may be analyzed to monitor the precision of the
sampling technique. When doubt exists over the identification of a peak on the chromatogram as
carbendazim, confirmatory techniques such as chromatography with a dissimilar column, or ratio
of absorbance at two or more wavelengths may be used. Whenever possible, the laboratory
should perform analysis of standard reference materials and participate in relevant performance
evaluation studies.
9. SAMPLE COLLECTION, PRESERVA nON, AND HANDLING
9.1 Grab samples must be collected in glass containers. Conventional sampling practices should be
followed; however, the bottle must not be prerinsed with sample before collection. Composite
samples should be collected in refrigerated glass containers in accordance with the requirements
of the program. Automatic sampling equipment must be as free as possible of plastic and other
potential sources of contamination.
9.2 The samples must be iced or refrigerated at 4°C from the time of collection until extraction.
9.3 All samples must be extracted within 7 days and completely analyzed within 40 days of extraction.
10. SAMPLE EXTRACTION
10.1 Using a 250-mL graduated cylinder; measure 150 mL of well-mixed sample into a 250-mL
Erlenmeyer flask. If benomyl is a potentiality in the sample, continue with Section 10.2. If only
carbendazim is to be measured, proceed directly to Section 10.3.
10.2 Carefully add 2 mL of 1+1 sulfuric acid and a TFE-fluorocarbon covered magnetic stirring bar
to the sample. Check the sample with wide-range pH paper to insure that the pH is less than 1.0.
Stir at room temperature for 16 to 24 hours.
10.3 Adjust the sample pH to within the range of 6 to 8 with sodium hydroxide. Pour the entire
sample into a 250-mL separatory funnel.
10.4 Add 60 mL methylene chloride to the separatory funnel and extract the sample by shaking the
funnel for 2 minutes with periodic venting to release excess pressure. Allow the organic layer to
separate from the water phase for a minimum of 10 minutes. If the emulsion interface between
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Method 631
layers is more than one-third the volume of the solvent layer, the analyst must employ mechanical
techniques to complete the phase separation. The optimum technique depends upon the sample,
but may include stirring, filtration of the emulsion through glass wool, centrifugation, or other
physical methods. Collect the methylene chloride extract in a 250-mL Erlenmeyer flask.
10.5 Add a second 60-mL volume of methylene chloride to the separatory funnel and repeat the
extraction procedure a second time, combining the extracts in the Erlenmeyer flask. Perform a
third extraction in the same manner.
10.6 Assemble a Kuderna-Danish (K-D) concentrator by attaching a 1O-mL concentrator tube to a
500-mL evaporative flask. Other concentration devices or techniques may be used in place of the
K-D if the requirements of Section 8.2 are met.
10.7 Pour the combined extract through a drying column containing about 10 cm of anhydrous sodium
sulfate, and collect the extract in the K-D concentrator. Rinse the Erlenmeyer flask and column
with 20 to 30 mL of methylene chloride to complete the quantitative transfer.
10.8 Add one or two clean boiling chips to the evaporative flask and attach a three-ball Snyder column.
Prewet the Snyder column by adding about 1 mL methylene chloride to the top. Place the K-D
apparatus on a hot water bath, 60 to 65°C, so that the concentrator tube is partially immersed in
the hot water, and the entire lower rounded surface of the flask is bathed with hot vapor. Adjust
the vertical position of the apparatus and the water temperature as required to complete the
concentration in 15 to 20 minutes. At the proper rate of distillation, the balls of the column will
actively chatter but the chambers will not flood with condensed solvent. When the apparent
volume of liquid reaches 1 mL, remove the K-D apparatus and allow it to drain and cool for at
least 10 minutes.
10.9 Increase the temperature of the hot water bath to 85 to 90°C. Momentarily remove the Snyder
column, add 50 mL of methanol and a new boiling chip and reattach the Snyder column. Pour
about 1 mL of methanol into the top of the Snyder column and concentrate the solvent extract as
before. Elapsed time of concentration should be 5 to 10 minutes. When the apparent volume of
liquid reaches I mL, remove the K-D apparatus and allow it to drain and cool for at least 10
minutes.
10.10 Remove the Snyder column and rinse the flask and its lower joint into the concentrator
tube with 1 to 2 mL of methanol and adjust the volume to 10 mL. A 5-mL syringe is
recommended for this operation. Stopper the concentrator tube and store refrigerated if
further processing will not be performed immediately. If the extracts will be stored
longer than 2 days, they should be transferred to TFE-fluorocarbon-sealed screw-cap
vials. Proceed with HPLC analysis.
11. CLEANUP AND SEPARA TION
11.1 Cleanup procedures may not be necessary for a relatively clean sample matrix. If particular
circumstances demand the use of a cleanup procedure, the analyst must determine the elution
profile and demonstrate that the recovery of each compound of interest for the cleanup procedure
is no lessthan 85%.
12. LiQuiD CHROMATOGRAPHY
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Method 631
12.1 Table 1 summarizes the recommended operating conditions for the liquid chromatograph.
Included in this table are the estimated retention time and method detection limit that can be
achieved by this method. An example of the separation achieved by this column is shown in
Figure 1. Other HPLC columns, chromatographic conditions, or detectors may be used if the
requirements of Section 8.2 are met.
12.2 Calibrate the system daily as described in Section 7.
12.3 If the internal standard approach is being used, add the internal standard to sample extracts
immediately before injection into the instrument. Mix thoroughly.
12.4 Inject 10 L of the sample extract. Record the volume injected to the nearest 0.05 L, and the
resulting peak size in area or peak height units.
12.5 The width of the retention-time window used to make identifications should be based upon
measurements of actual retention-time variations of standards over the course of a day. Three.
times the standard deviation of a retention time can be used to calculate a suggested window size
for a compound. However, the experience of the analyst should weigh heavily in the
interpretation of chromatograms.
12.6 If the response for the peak exceeds the working range of the system, dilute the extract and
reanalyze.
12.7 If the measurement of the peak response is prevented by the presence of interferences, further
cleanup is required.
13. CAL CL/LA TIONS
13.1 Determine the concentration of carbendazim in the sample.
13.1.1 If the external standard calibration procedure is used, calculate the amount of material
injected from the peak response using the calibration curve or calibration factor in
Section 7.2.2. The concentration in the sample can be calculated as follows:
Equation 2
( A)(V )
Concentration, iiglL = (V )(V)
where
A = Amount of material injected, in ng
V , = Voiwne of extract injected, in pL
= Volume of total extract, in uL
V 5 = Volume of water extracted, in mL
13.1.2 If the internal standard calibration procedure was used, calculate the concentration in the
sample using the response factor (RF) determined in Section 7.3.2 as follows:
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Method 631
Equation 3
Concentration, igIL = S $
(A )(RF)(V 0 )
where
A 5 = Response for parameter to be measured
A = Response for the internal standard
I , = Amount of internal standard added to each extract, in ig
V 0 = Volume of water extracted, in L
13.2 If the sample was treated to hydrolyze benomyl, report the results as benomyl (measured as
carbendazim). If the hydrolysis step was omitted, report results as carbendazim. Report results
in micrograms per liter without correction for recovery data. When duplicate and spiked samples
are analyzed, report all data obtained with the sample results.
13.3 For samples processed as part of a set where the laboratory spiked sample recovery falls outside
of the control limits in Section 8.3, data for the affected parameters must be labeled as suspect.
14. METHOD PERFORMANCE -
14.1 The method detection limit (MDL) is defined as the minimum concentration of a substance that
can be measured and reported with 99% confidence that the value is above zero. 9 The MDL
concentrations listed in Table 1 were determined by extracting 1000-mL aliquots of reagent water
with three 350 mL volumes of methylene chloride.’
14.2 In a single laboratory (West Cost Technical Services, Inc.), using reagent water and effluents from
publicly owned treatment works (POTW), the average recoveries presented in Table 2 were
obtained.’ The standard deviations of the percent recoveries of these measurements are also
included in Table 2. All results were obtained using the same experimental scale described in
Section 14.1.
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Method 631
References
1. “Pesticide Methods Evaluation,” Letter Report #17 for EPA Contract No. 68-03-2697. Available
from U.S. Environmental Protection Agency, Environmental Monitoring and Support Laboratory,
Cincinnati, Ohio.
2. “Development of Analytical Test Procedures for Organic Pollutants in Wastewater-Application to
Pesticides,” EPA Report 600/4-81-017, U.S. Environmental Protection Agency, Cincinnati, Ohio
45268. PB #82 132507, National Technical Information Service, Springfield, Virginia.
3. ASTM Annual Book of Standards, Part 31, D3694, “Standard Practice for Preparation of Sample
Containers and for Preservation, American Society for Testing and Materials, Philadelphia,
Pennsylvania, p. 679, 1980.
4. “Carcinogens - Working with Carcinogens,” Department of Health, Education, and Welfare,
Public Health Service, Center for Disease Control, National Institute for Occupational Safety and
Health, Publication No. 77-206, Aug. 1977.
5. “OSHA Safety and Health Standards, General Industry,” (29 CFR 1910), Occupational Safety and
Health Administration, OSHA 2206, (Revised, January 1976).
6. “Safety in Academic Chemistry Laboratories,” American Chemical Society Publication,
Committee on Chemical Safety, 3rd Edition, 1979.
7. “Handbook for Analytical Quality Control in Water and Wastewater Laboratories,”
EPA-600/4-79-019, U.S. Environmental Protection Agency, Environmental Monitoring and
Support Laboratory, Cincinnati, Ohio, March 1979.
8. ASTM Annual Book of Standards, Part 31, D3370, “Standard Practice for Sampling Water,”
American Society for Testing and Materials, Philadelphia, Pennsylvania, p. 76, 1980.
9. Glaser, J.A. et.al, “Trace Analysis for Wastewaters,” Environmental Science & Technology, 15,
1426 (1981).
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Method 631
Table 1. Chromatographic Conditions and Method Detection Limits
Retention Time Method Detection Limit (pg/LI
Parameter (mm)
Benomyl (as carbendazim) 25.0
Carbendazim 8.1 8.7
Column conditions: Bondapak C 18 (10 inn) packed in a 30 cm long by 4 mm ID stainless steel column
with a mobile phase flow rate of 2.0 mL/min at ambient temperature.
Mobile phase: methanol/water (1+1).
Table 2. Single-Operator Accuracy and Precision
Number Average Standard
Sample of Spike Percent Deviation
Parameter Type Replicates (pg/LI Recovery (96)
DW 7 51.5 70 15.5
Benomyl (as carbendazim) MW 7 51.5 78 8.8
MW 7 103 99 6.4
Carbendazim DW 7 50 106 5.5
MW 7 50 117 18.5
MW 7 100 108 11.3
DW = Reagent water
MW = Municipal wastewater
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Method 631
Carbindazim
- ‘
0 10
Retention Time (minutes)
A5200242
Figure 1. Liquid Chromatogram of Carbendazim on Column 1.
For Conditions, See Table 1.
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Method 632
The Determination of
Carbamate and Urea Pesticides
in Municipal and Industrial
Waste water
-------
Method 632
The Determination of Carbamate and Urea Pesticides in Municipal
and Industrial Wastewater
SCOPE AND APPLiCATION
1 .1 This method covers the determination of certain carbamate and urea pesticides. The following
parameters can be determined by this method:
Parameter STORET No. CAS No.
Aminocarb — 2032-59-9
Barban — 101-27-9
Carbaryl 39750 63-25-2
Carbofuran 81405 1563-66-2
Chiorpropham — 101-21-3
Diuron 39650 330-54-1
Fenuron — 101-42-8
Fenuron-TCA — 4482-55-7
Fluometuron 2164-17-2
Linuron 330-55-2
Methiocarb — 2032-65-7
Methomyl 39051 16752-77-5
Mexacarbate — 315-18-4
Monuron 150-68-5
Monuron-TCA 14041-0
Neburon 555-37-3
Oxamyl — 23135220
Propham 39052 122-42-9
Propoxur 114-26-1
Siduron 198249-6
Swep 1918-18-9
1.2 This method cannot distinguish rnonuron from monuron-TCA and fenuron from fenuron-TCA.
Results for the paired parameters are reported as monuron and fenuron respectively.
1.3 This is a high performance liquid chromatographic (IIPLC) method applicable to the determination
of the compounds listed above in industrial and municipal discharges as provided under 40 CFR
136.1. Any modification of this method beyond those expressly permitted shall be considered a
major modification subject to application and approval of alternative test procedures under
40 CFR 136.4 and 136.5.
1.4 The method detection limit (MDL, defined in Section 15) for many of the parameters are listed
in Table 1. The MDL for a specific wastewater may differ from those listed, depending upon the
nature of interferences in the sample matrix.
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Method 632
1.5 This method is restricted to use by or under the supervision of analysts experienced in the use of
liquid chromatography and in the interpretation of liquid chromatograms. Each analyst must
demonstrate the ability to generate acceptable results with this method using the procedure
described in Section 8.2.
1.6 When this method is used to analyze unfamiliar samples for any or all of the compounds above,
compound identifications should be supported by at least one additional qualitative technique.
2. SUMMARY OF METHOD
2.1 A measured volume of sample, approximately 1 L, is extracted with methylene chloride using a
separatory funnel. The methylene chloride extract is dried and concentrated to a volume of 10
mL or less. HPLC chromatographic conditions are described which permit the separation and
measurement of the compounds in the extract by HPLC with a UV detector. 1 2
2.2 This method provides an optional Florisil column cleanup procedure to aid in the elimination or
reduction of interferences which may be encountered.
3. INTERFERENCES
3.1 Method interferences may be caused by contaminants in solvents, reagents, glassware, and other
sample-processing apparatus that lead to discrete artifacts or elevated baselines in liquid chromato-
grams. All reagents and apparatus must be routinely demonstrated to be free from interferences
under the conditions of the analysis by running laboratory reagent blanks as described in
Section 8.5.
3.1.1 Glassware must be scrupulously cleaned. 3 Clean all glassware, as soon as possible after
use by thoroughly rinsing with the last solvent used in it. Follow by washing with hot
water and detergent and thorough rinsing with tap and reagent water. Drain dry, and
heat in an oven or muffle furnace at 400°C for 15 to 30 minutes. Do not heat volumetric
ware. Thermally stable materials, such as PCBs, may not be eliminated by this
treatment. Thorough rinsing with acetone and pesticide-quality hexane may be substituted
for the heating. After drying and cooling, seal and store glassware in a clean
environment to prevent any accumulation of dust or other contaminants. Store inverted
or capped with aluminum foil.
3.1.2 The use of high-purity reagents and solvents helps to minimize interference problems.
Purification of solvents by distillation in all-glass systems may be required.
3.2 Matrix interferences may be caused by contaminants that are coextracted from the sample. The
extent of matrix interferences will vary considerably from source to source, depending upon the
nature and diversity of the industrial complex or municipality sampled. The cleanup procedure
in Section 11 can be used to overcome many of these interferences, but unique samples may
require additional cleanup approaches to achieve the MDL listed in Table 1.
4. SAFETY
4.1 The toxicity or carcinogenicity of each reagent used in this method has not been precisely defined;
however, each chemical compound must be treated as a potential health hazard. From this
viewpoint, exposure to these chemicals must be reduced to the lowest possible level by whatever
350
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Method 632
means available. The laboratory is responsible for maintaining a current awareness file of OSHA
regulations regarding the safe handling of the chemicals specified in this method. A reference file
of material data handling sheets should also be made available to all personnel involved in the
chemical analysis. Additional references to laboratory safety are available and have been
identified for the information of the analyst.
5. APPARA TUS AND MA TERIALS
5.1 Sampling equipment, for discrete or composite sampling.
5.1 .1 Grap-sample bottle: Amber borosilicate or flint glass, 1-L or 1-quart volume, fitted with
screw-caps lined with TFE-fluorocarbon. Aluminum foil may be substituted for TFE if
the sample is not corrosive. If amber bottles are not available, protect samples from
light. The container and cap liner must be washed, rinsed with acetone or methylene
chloride, and dried before use to minimize contamination.
5.1 .2 Automatic sampler (optional): Must incorporate glass sample containers for the
collection of a minimum of 250 mL. Sample containers must be kept refrigerated at 4°C
and protected from light during compositing. If the sampler uses a peristaltic pump, a
minimum length of compressible silicone rubber tubing may be used. Before use,
however, the compressible tubing must be thoroughly rinsed with methanol, followed by
repeated rinsings with reagent water to minimize the potential for contamination of the
sample. An integrating flow meter is required to collect flow-proportional composites.
5.2 Glassware. (All specifications are suggested. Catalog numbers are included for illustration only.)
5.2.1 Separatory funnel 2000-mL, with TFE-fluorocarbon stopcock, ground-glass or TFE
stopper.
5.2.2 Drying column: Chromatographic column 400 mm long by 19 mm ID with coarse-fitted
disc.
5.2.3 Chromatographic column: 400 mm long by 19 mm ID with coarse-fitted disc at bottom
and TFE-fluorocarbon stopcock (Kontes K-420540-0224 or equivalent).
5.2.4 Flask: round-bottom 500-mL, with standard taper to fit rotary evaporator.
5.2.5 Vials: Amber glass, 10- to 15-mL capacity with TFE-fluorocarbon-lined screw-cap.
5.3 Rotary evaporator.
5.4 Water bath: Heated, with concentric ring cover, capable of temperature control (± 2°C). The
bath should be used in a hood.
5.5 Balance: Analytical, capable of accurately weighing to the nearest 0.0001 g.
5.6 Filtration apparatus: As needed to filter chromatographic solvents prior to HPLC.
5.7 Liquid chromatograph: High performance analytical system complete with high pressure syringes
or sample injection loop, analytical columns, detector, and strip-chart recorder. A guard column
is recommended for all applications.
5.7.1 Gradient pumping system, constant flow.
5.7.2 Column: 30 cm long by 4 mm ID stainless steel packed with L Bondapak C 18 (10 m)
or equivalent. This column was used to develop the method performance statements in
351
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Method 632
Section 14. Alternative columns may be used in accordance with the provisions
described in Section 12.1.
5.7.3 Detector: Ultraviolet, capable of monitoring at 254 nm and 280 nm. This detector has
proven effective in the analysis of wastewaters and was used to develop the method
performance statements in Section 14. Alternative detectors may be used in accordance
with the provisions described in Section 12.1.
6. REAGENTS
6.1 Reagent water: Reagent water is defined as a water in which an interferent is not observed at the
method detection limit of each parameter of interest.
6.2 Acetone, acetonitrile, hexane, methylene chloride, methanol: Pesticide-quality or equivalent.
6.3 Ethyl ether: Nanograde, redistilled in glass if necessary. Must be free of peroxides as indicated
by EM Quant test strips (available from Scientific Products Co., Cat. No. P1126-8, and other
suppliers). Procedures recommended for removal of peroxides are provided with the test strips.
After cleanup, 20 mL ethyl alcohol preservative must be added to each liter of ether.
6.4 Sodium sulfate: ACS, granular, anhydrous. Condition by heating in a shallow tray at 400°C for
a minimum of 4 hours to remove phthalates and other interfering organic substances.
Alternatively, heat 16 hours at 450 to 500°C in a shallow tray or perform a Soxhiet extraction
with methylene chloride for 48 hours.
6.5 Florisil: PR grade (60/100 mesh). Purchase activated at 675°C and store in dark in glass
container with ground-glass stopper or foil-lined screw-cap. Before use activate each batch at least
16 hours at 130°C in a foil-covered glass container.
6.6 Acetic acid: Glacial.
6.7 Stock standard solutions (1.00 gI . L): Stock standard solutions may be prepared from pure
standard materials or purchased as certified solutions.
6.1.1 Prepare stock standard solutions by accurately weighing approximately 0.0100 g of pure
material. Dissolve the material in pesticide-quality acetonitrile or methanol and dilute to
volume in a l0-mL volumetric flask. Larger volumes may be used at the convenience
of the analyst. If compound purity is certified at 96% or greater, the weight may be used
without correction to calculate the concentration of the stock standard. Commercially
prepared stock standards may be used at any concentration if they are certified by the
manufacturer or by an independent source.
6.7.2 Transfer the stock standard solutions into TFE-fluorocarbon-sealed screw-cap vials.
Store at 4°C and protect from light. Frequently check stock standard solutions for signs
of degradation or evaporation, especially just prior to preparing calibration standards
from them.
6.7.3 Stock standard solutions must be replaced after 6 months, or sooner if comparison with
check standards indicates a problem.
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Method 632
7. CAL/BRA TION
7.1 Establish HPLC operating parameters equivalent to those indicated in Table 1. The HPLC system
may be calibrated using either the external standard technique (Section 7.2) or the internal
standard technique (Section 7.3).
For each parameter of interest, prepare calibration standards at a minimum of three
concentration levels by adding accurately measured volumes of one or more stock
standards to a volumetric flask and diluting to volume with acetonitrile or methanol. One
of the external standards should be representative of a concentration near, but above, the
method detection limit. The other concentrations should correspond to the range of
concentrations expected in the sample concentrates or should define the working range
of the detector.
7.2.2 Using injections of 10 j L of each calibration standard, tabulate peak height or area
responses against the mass injected. The results can be used to prepare a calibration
curve for each parameter. Alternatively, the ratio of the response to the mass Injected,
defined as the calibration factor (CF), may be calculated for each parameter at each
standard concentration. If the relative standard deviation of the calibration factor is less
than 10% over the working range, the average calibration factor can be used in place of
a calibration curve.
7.2.3 The working calibration curve or calibration factor must be verified on each working
shift by the measurement of one or more calibration standards. If the response for any
parameter varies from the predicted response by more than ± 10%, the test must be
repeated using a fresh calibration standard. Alternatively, a new calibration curve or
calibration factor must be prepared for that parameter.
7.3 Internal standard calibration procedure: To use this approach, the analyst must select one or more
internal standards similar in analytical behavior to the compounds of interest. The analyst must
further demonstrate that the measurement of the internal standard is not affected by method or
matrix interferences. Due to these limitations, no internal standard applicable to all samples can
be suggested.
7.3.1 Prepare calibration standards at a minimum of three concentration levels for each
parameter of interest by adding volumes of one or more stock standards to a volumetric
flask. To each calibration standard, add a known constant amount of one or more
internal standards, and dilute to volume with acetonitrile or methanol. One of the
standards should be representative of a concentration near, but above, the method
detection limit. The other concentrations should correspond to the range of
concentrations expected in the sample concentrates, or should define the working range
of the detector.
7.3.2 Using injections of 10 1 zL of each calibration standard, tabulate the peak height or area
responses against the concentration for each compound and internal standard. Calculate
response factors (RF) for each compound as follows:
7.2 External standard calibration procedure.
7.2.1
353
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Method 632
Equation 1
= ( A,)(C )
(A )(C,)
where
A 3 = Response for the parameter to be measured
A , = Response for the internal standard
C = Concentration of the internal standard, in g/L
C = Concentration of the parameter to be measured, in j. gIL
If the RF value over the working range is constant, less than 10% relative standard
deviation, the RF can be assumed to be invariant and the average RF may be used for
calculations. Alternatively, the results may be used to plot a calibration curve of
response ratios, A /A against RF.
7.3.3 The working calibration curve or RF must be verified on each working shift by the
measurement of one or more calibration standards. If the response for any parameter
varies from the predicted response by more than ± 10%, the test must be repeated using
a fresh calibration standard. Alternatively, a new calibration curve must be prepared for
that compound.
7.4 The cleanup procedure in Section 11 utilizes Florisil chromatography. Florisil from different
batches or sources may vary in adsorptive capacity. To standardize the amount of Florisil which
is used, the use of the laurie acid value is suggested. This procedure 7 determines the adsorption
from hexane solution of the laurie acid, in milligrams per gram of Florisil. The amount of
Florisil to be used for each column is calculated by dividing this factor into 110 and multiplying
by 20 g.
7.5 Before using any cleanup procedure, the analyst must process a series of calibration standards
through the procedure to validate elution patterns and the absence of interference from the
reagents.
8. QUALITY CONTROL
8.1 Each laboratory using this method is required to operate a formal quality control program. The
minimum requirements of this program consist of an initial demonstration of laboratory capability
and the analysis of spiked samples as a continuing check on performance. The laboratory is
required to maintain performance records to define the quality of data that is generated.
8.1.1 Before performing any analyses, the analyst must demonstrate the ability to generate
acceptable accuracy and precision with this method. This ability is established as
described in Section 8.2.
8.1.2 In recognition of the rapid advances occurring in chromatography, the analyst is
permitted certain options to improve the separations or lower the cost of measurements.
Each time such modifications to the method are made, the analyst is required to repeat
the procedure in Section 8.2.
8.1.3 The laboratory must spike and analyze a minimum of 10% of all samples to monitor
continuing laboratory performance. This procedure is described in Section 8.4.
354
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Method 632
8.2 To establish the ability to generate acceptable accuracy and precision, the analyst must perform
the following operations.
8.2.1 Select a representative spike concentration for each compound to be measured; Using
stock standards, prepare a quality control check sample concentrate in acetonitrile or
methanol, 1000 times more concentrated than the selected concentrations.
8.2.2 Using a pipette, add 1.00 mL of the check sample concentrate to each of a minimum of
four 1000-mi. aliquots of reagent water. A representative wastewater may be used in
place of the reagent water, but one or more additional aliquots must be analyzed to
determine background levels, and the spike level must exceed twice the background level
for the test to be valid. Analyze the aliquots according to the method beginning in
Section 10.
8.2.3 Calculate the average percent recovery (R), and the standard deviation of the percent
recovery (s), for the results. Wastewater background corrections must be made before
R and s calculations are performed.
8.2.4 Table 2 provides single-operator recovery and precision for most of the carbamate and
urea pesticides. Similar results should be expected from reagent water for all compounds
listed in the method. Compare these results to the values calculated in Section 8.2.3.
If the data are not comparable, review potential problem areas and repeat the test.
8.3 The analyst must calculate method performance criteria and define the performance of: the
laboratory for each spike concentration and parameter being measured.
8.3.1 Calculate upper and lower control limits for method performance as follows:
Upper Control Limit (UCL) = R + 3s
Lower Control Limit (LCL) = R - 3s
where R and s are calculated as in Section 8.2.3. The UCL and LCL can be used to
construct control charts 8 that are useful in observing trends in performance.
8.3.2 The laboratory must develop and maintain separate accuracy statements of laboratory
performance for wastewater samples. An accuracy statement for the method is defined
as R ± s. The accuracy statement should be developed by the analysis of four aliquots
of wastewater as described in Section 8.2.2, followed by the calculation of R and s.
Alternatively, the analyst may use four wastewater data points gathered through the
requirement for continuing quality control in Section 8.4. The accuracy statements
should be updated regularly. 8
8.4 The laboratory is required to collect in duplicate a portion of their samples to monitor spike
recoveries. The frequency of spiked sample analysis must be at least 10% of all samples or one
spiked sample per month, whichever is greater. One aliquot of the sample must be spiked and
analyzed as described in Section 8.2. If the recovery for a particular parameter does not fall
within the control limits for method performance, the results reported for that parameter in all
samples processed as part of the same set must be qualified as described in Section 13.3. The
laboratory should monitor the frequency of data so qualified to ensure that it remains at or
below 5%.
355
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Method 632
8.5 Before processing any samples, the analyst must demonstrate through the analysis of a l-L aliquot
of reagent water that all glassware and reagent interferences are under control. Each time a set
of samples is extracted or there is a change in reagents, a laboratory reagent blank must be
processed as a safeguard against laboratory contamination.
8.6 It is recommended that the laboratory adopt additional quality assurance practices for use with this
method. The specific practices that are most productive depend upon the needs of the laboratory
and the nature of the samples. Field duplicates may be analyzed to monitor the precision of the
sampling technique. When doubt exists over the identification of a peak on the chromatogram,
confirmatory techniques, such as chromatography with a dissimilar column, or ratio of absorbance
at two or more wavelengths, may be used. Whenever possible, the laboratory should perform
analysis of quality control materials and participate in relevant performance evaluation studies.
9. SAMPLE COLLECTION, PRESERVATiON, AND HANDLING
9.1 Grap-samples must be collected in glass containers. Conventional sampling practices 9 should be
followed; however, the bottle must not be prerinsed with sample before collection. Composite
samples should be collected in refrigerated glass containers in accordance with the requirements
of the program. Automatic sampling equipment must be as free as possible of plastic and other
potential sources of contamination.
9.2 The samples must be iced or refrigerated at 4°C from the time of collection until extraction.
9.3 All samples must be extracted within 7 days and completely analyzed within 40 days of extraction.
10. SAMPLE EXTRACTION
10.1 Mark the water meniscus on the side of the sample bottle for later determination of sample
volume. Pour the entire sample into a 2-L separatory funnel.
10.2 Add 60 mL methylene chloride to the sample bottle, seal, and shake 30 seconds to rinse the inner
walls. Transfer the solvent to the separatory funnel and extract the sample by shaking the funnel
for 2 minutes with periodic venting to release excess pressure. Allow the organic layer to separate
from the water phase for a minimum of 10 minutes. If the emulsion interface between layers is
more than one-third the volume of the solvent layer, the analyst must employ mechanical
techniques to complete the phase separation. The optimum technique depends upon the sample,
but may include stirring, filtration of the emulsion through glass wool, centrifugation, or other
physical methods. Collect the methylene chloride extract in a 250-mL Erlenineyer flask.
10.3 Add a second 60-mL volume of methylene chloride to the sample bottle and repeat the extraction
procedure a second time, combining the extracts in the Erlenmeyer flask. Perform a third
extraction in the same manner.
10.4 It is necessary to exchange the extract solvent to hexane if the Florisil clean up procedure is to
be used. For direct HPLC analysis the extract solvent must be exchanged to a solvent (either
methanol or acetonitrile) that is compatible with the mobile phase. The analyst should only
exchange a portion of the extract to HPLC solvent if there is a possibility that cleanup may be
necessary.
356
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Method 632
10.5 Pass a measured fraction or all of the combined extract through a drying column containing about
10 cm of anhydrous sodium sulfate and collect the extract in a 500-mL round-bottom flask. Rinse
the Erlenmeyer flask and column with 20 to 30 mL of methylene chloride to complete the
quantitative transfer.
10.6 Attach the 500-mL round-bottom flask containing the extract to the rotary evaporator and partially
immerse in the 50°C water bath.
10.7 Concentrate the extract to approximately 5 mL in the rotary evaporator at a temperature of 50°C.
Other concentration techniques may be used if the requirements of Section 8.2 are met.
10.8 Add 50 inL of hexane, methanol, or acetonitrile to the round-bottom flask and concentrate the
solvent extract as before. When the apparent volume of liquid reaches approximately 5 mL
remove the 500-mL round-bottom flask from the rotary evaporator and transfer the concentrated
extract to a 10-mL volumetric flask, quantitatively washing with 2 mL of solvent. Adjust the
volume to 10 mL.
10.9 Stopper the volumetric flask and store refrigerated at 4°C if further processing will not be
performed immediately. If the extracts will be stored longer than 2 days, they should be
transferred to TFE-fluorocarbon-sealed screw-cap bottles.
10.10 Determine the original sample volume by refilling the sample bottle to the mark and transferring
the water to a 1000-mL graduated cylinder. Record the sample volume to the nearest 5 mL.
11. CLEANUP AND SEPARA TION
11.1 Cleanup procedures may not be necessary for a relatively clean sample matrix. The cleanup
procedure recommended in this method has been used for the analysis of various industrial and
municipal effluents. If particular circumstances demand the use of an alternative cleanup
procedure, the analyst must determine the elution profile and demonstrate that the recovery of
each compound of interest for the cleanup procedure is no less than 85%.
11.2 The following Florisil column cleanup procedure has been demonstrated to be applicable to the
five pesticides listed in Table 3. It should also be applicable to the cleanup of extracts for the
other carbamate and urea pesticides listed in the scope of this method.
11.2.1 Add a weight of Florisil (nominally 20 g) predetermined by calibration (Section 7.4
and 7.5), to a chromatographic column. Settle the Florisil by tapping the column. Add
anhydrous sodium sulfate to the top of the Florisil to form a layer 1 to 2 cm deep. Add
60 mL of hexane to wet and rinse the sodium sulfate and Florisil. Just prior to exposure
of the sodium sulfate to air, stop the elution of the hexane by closing the stopcock on the
chromatography column. Discard the eluate.
11.2.2 Adjust the sample extract volume to 10 mL with hexane and transfer it from the
volumetric flask to the Florisil column. Rinse the flask twice with 1 to 2 mL hexane,
adding each rinse to the column.
1 1.2.3 Drain the column until the sodium sulfate layer is nearly exposed. Elute the column with
200 mL of 20% ethyl ether in hexane (VIV) (Fraction 1) using a drip rate of about
5 mL/min. Place a 500-mL round-bottom flask under the chromatography column.
Elute the column again, using 200 mL of 6% acetone in hexane (V/V) (Fraction 2), into
a second flask. Perform a third elution using 200 mL of 15% acetone in hexane (V/V)
357
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Method 632
(Fraction 3), and a final elution with 200 mL of 50% acetone in hexane (V/V)
(Fraction 4), into separate flasks. The elution patterns for five of the pesticides are
shown in Table 3.
11.2.4 Concentrate the eluates to 10 mL with a rotary evaporator as described in Section 10.7,
exchanging the solvent to acetonitrile or methanol as required.
12. LIQuID CHROMATOGRAPHY
12.1 Table 1 summarizes the recommended operating conditions for the liquid chromatograph.
Included in this table are estimated retention-times and method detection limits that can be
achieved by this method. An example of the separations achieved by this column is shown in
Figure 1. Other HPLC columns, chromatographic conditions, or detectors may be used if the
requirements of Section 8.2 are met.
12.2 Calibrate the system daily as described in Section 7. The standards and extracts must be in the
solvent (acetonitrile or methanol) compatible with the mobile phase.
12.3 If the internal standard approach is being used, add the internal standard to sample extracts
immediately before injection into the instrument. Mix thoroughly.
12.4 Inject 10 L of the sample extract. Record the volume injected to the nearest 0.05 L, and the
resulting peak size in area or peak height units.
12.5 The width of the retention-time window used to make identifications should be based upon
measurements of actual retention-time variations of standards over the course of a day. Three
times the standard deviation of a retention time can be used to calculate a suggested window size
for a compound. However, the experience of the analyst should weigh heavily in the
interpretation of chromatograms.
12.6 If the response for the peak exceeds the working range of the system, dilute the extract and
reanalyze.
12.7 If the measurement of the peak response is prevented by the presence of interferences, further
cleanup is required.
13. CALCULATIONS
13.1 Determine the concentration of individual compounds in the sample.
13.1.1 If the external standard calibration procedure is used, calculate the amount of material
injected from the peak response using the calibration curve or calibration factor in
Section 7.2.2. The concentration in the sample can be calculated as follows:
358
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Method 632
Equation 2
Concentration, ,LgIL = (A)(V)
(V 1 )(V,)
where
A = Amount of material injected, in ng
V. = Voiwne of extract injected, in pL
V 1 = Voiwne of total extract, in pL
= Volume of water extracted, in mL
13.1.2 If the internal standard calibration procedure was used, calculate the concentration in the
sample using the response factor (RE) determined in Section 7.3.2 as follows:
Equation 3
(A,)(I,)
Concentration, 4 ugIL = (A )(RF)(V 0 )
where
A =
Response for parameter to be measured
A =
Response for the internal standard
=
Amount of internal standard added to each extract,
in
g
V 0 =
Volume of water extracted, in L
13.2 Calculate and report fenuron-TCA as femiron and monuron-TCA as monuron. Report results in
micrograms per liter without correction for recovery data. When duplicate and spiked samples
are analyzed, report all data obtained with the sample results.
13.3 For samples processed as part of a set where the laboratory spiked sample recovery falls outside
of the control limits in Section 8.3, data for the affected parameters must be labeled as suspect.
14. METHOD PERFORMANCE
14.1 The method detection limit (MDL) is defined as the minimum concentration of a substance that
can be measured and reported with 99% confidence that the value is above zero.’° The MDL
concentrations listed in Table I were obtained using reagent water or river water. 2 ’ 11
14.2 In a single laboratory, the average recoveries presented in Table 2 were obtained using this
method. 2’1 ’ The standard deviations of the percent recoveries of these measurements are also
included in Table 2.
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Method 632
References
1. “Development of Analytical Test Procedures for Organic Pollutants in Wastewater-Application to
Pesticides,” EPA Report 600/4-81-017, U.S. Environmental Protection Agency, Cincinnati, Ohio
45268. PB#82 132507, National Technical Information Service, Springfield, Va.
2. Farrington, D.S., Hopkins, R.G. and Ruzicka, JH.A. “Determination of Residues of Substituted
Phenylurea Herbicides in Grain, Soil, and River Water by Use of Liquid Chromatography,”
Analyst, 102, 377-38 1 (1977).
3. ASTM Annual Book of Standards, Part 31, D3694, “Standard Practice for Preparation of Sample
Containers and for Preservation, “American Society for Testing and Materials, Philadelphia, PA,
p. 679, 1980.
4. “Carcinogens - Working with Carcinogens,” Department of Health, Education, and Welfare,
Public Health Service, Center for Disease Control, National Institute for Occupational Safety and
Health, Publication No. 77-206, Aug. 1977.
5. “OSHA Safety and Health Standards, General Industry,” (29 CFR 1910), Occupational Safety and
Health Administration, OSHA 2206, (Revised, January 1976).
6. “Safety in Academic Chemistry Laboratories,” American Chemical Society Publication,
Committee on Chemical Safety, 3rd Edition, 1979.
7. ASTM Annual Book of Standards, Part 31, D3086, Appendix X3, “Standardization of Florisil
Column by Weight Adjustment Based on Adsorption of Lauric Acid,” American Society for
Testing and Materials, Philadelphia, PA, p 765, 1980.
8. “Handbook for Analytical Quality Control in Water and Wastewater Laboratories,”
EPA-600/4-79 -019, U.S. Environmental Protection Agency, Environmental Monitoring and
Support Laboratory - Cincinnati, Ohio 45268, March 1979.
9. ASTM Annual Book of Standards, Part 31, D3370, “Standard Practice for Sampling Water,”
American Society for Testing and Materials, Philadelphia, PA, p. 76, 1980.
10. Glaser, J.A. et al., “Trace Analysis for Wastewaters,” Environmental Science & Technology, 15,
1426 (1981).
11. “Pesticide Methods Evaluation,” Letter Reports #12B, 18, 19, 20, 22 and 23 for EPA Contract
No. 68-03-2697. Available from U.S. Environmental Protection Agency, Environmental
Monitoring and Support Laboratory, Cincinnati, Ohio 45268.
360
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Method 632
Table 1. Chromatographic Conditions and Method Detection Limits
Method
UV Wavelength Detection
Mobile Retention (nm) Limit
Parameter Phase * Time (Mini (pg/Li
Mexacarbate A 8.7 254 0.52
Propoxur A 14.3 280 0.11
Monuron A 14.4 254 0.003
Carbaryl A 17.0 280 0.02
Propham A 17.2 254 0.07
Diuron A 19.5 254 0.009
Linuron A 21.0 254 0.009
Methiocarb A 21.4 254 0.02
Chiorprophani A 21.8 254 0.03
Barban A 22.3 254 0.05
Neburon A 24.3 254 0.012
Propoxur B 2.0 280 0.11
Methomyl B 6.5 254 8.9
Carbaryl B 14.1 280 0.02
Diuron B 15.5 254 0.009
Linuron B 17.9 254 0.009
Propoxur C 1.7 280 0.11
Carbofuran C 3.5 280 3.2
Fluorometuron C 3.6 254 11.1
Oxamyl D 3.2 254 9.2
*Mobjle Phase:
A Methanol/i % acetic acid, programmed linearly from 5 to 95% methanol at 2.0 mL/min flow rate
and at ambient temperature.
B Acetonitrile/water, programmed linearly from 10% to 100% acetonitrile in 30 minutes at a flow
rate of 2.0 mLlmin.
C 50% acetonitrile in water at a flow rate of 2.0 mL/min.
D 35% methanol in water at a flow rate of 2.0 mL/min.
Column: Bondapak C 18 (10 im) packed in a 30 cm long by 4 mm ID stainless steel column, with a
Whatmann Co. PELL ODS (30-38 &m) guard column, 7 cm long by 4 mm ID.
361
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Method 632
Table 2. Single-Operator Accuracy and Precision
Average Standard
Sample Spike No. of Percent Deviation
Parameter Type* (pg/U Analyses Recovery
Fluorometuron 1 50 7 93.9 7.0
2 50 7 80.0 7.2
4 1724 7 99 11.6
Propoxur 1 550 7 94.5 1.7
3 2200 3 105 3.0
4 550 7 87.2 7.3
5 0.5 5 93 6.0
Oxamyl 1 100 7 87 8.4
2 53 7 84.9 5.5
2 1080 7 89.8 2.7
Methomyl 1 100 4 74.4 2.4
3 30660 4 48.2 2.8
2 100 7 91.8 2.8
2 1960 7 94.4 1.9
Diuron 1 10 4 89.8 1.0
3 500 4 56.1 5.0
2 10 7 90.0 2.5
2 400 7 95.7 3.2
5 0.05 5 98 4.7
Linuron 1 10 4 95.0 3.4
3 4000 4 72.2 5.1
2 10 7 93.0 1.5
2 210 7 103 4.6
5 0.05 5 99 4.7
Carbofuran 1 37 7 87.8 2.7
4 148 7 99.3 1.4
Barban 5 0.3 5 98 4.1
Carbaryl 5 0.1 5 101 4.1
Chlorpropham 5 0.2 5 95 3.9
Methiocarb 5 0.2 5 95 2.6
Mexacarbate 5 4.0 5 96 3.5
Monuron 5 0.05 5 97 1.7
Neburon 5 0.05 5 96 6.6
Propham 5 0.3 5 88 5.9
* Sample Type
I = Reagent Water
2 = Municipal wastewater
3 = Industrial process water, pesticide manufacturing
4 = Industrial wastewater, pesticide manufacturing
5 = River Water
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Method 632
Tab’e 3. FIorisil Fractionation Patterns
Percent Recovery by Fraction
Parameter No. I No. 2 No. 3 No. 4
Diuron 0 0 24 58
Linuron 0 13 82 0
Methomyl 0 0 0 84
Oxamyl 0 0 92 0
Propachlor 0 94 0 0
Florisil eluate composition by fraction:
Fraction 1 - 200 mL of 20% ethyl ether in hexane
Fraction 2 - 200 mL of 6% acetone in hexane
Fraction 3 - 200 mL of 15% acetone in hexane
Fraction 4 - 200 mL of 50% acetone in hexane
363
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Method 632
Diuron
Methomyl /
/
Linuron
/
I I I I
0 5.0 10.0 15.0 200
Retention Time (minutes)
A52 63
Figure 1. Liquid Chromatogram of Diuron, Unuron and Methomyl on Column 1.
For Conditions, See Table 1.
364
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Method 632.1
The Determination of
Carbamate and Amide
Pesticides in Municipal and
Industrial Waste water
-------
Method 632.1
The Determination of Carbamate and Amide Pesticides
in Municipal and Industrial Waste water
1. SCOPE AND APPLICATiON
1 .1 This method covers the determination of certain carbarnatelamide pesticides in municipal and
industrial wastewater. The following parameters may be determined by this method.
Parameter CAS No.
Napropamide 15299-99-1
Propanil 709-98-8
Vacor 53558-25-1
1.2 The estimated detection limits (EDLs) for the parameters above are listed in Table 1. The EDL
was calculated from the minimum detectable response being equal to five times the background
noise using a 1O-mL final extract volume of a 1-L sample and an injection volume of 100 iL.
The EDL for a specific wastewater may be different depending on the nature of interferences in
the sample matrix.
1 .3 This is a high performance liquid chromatographic (HPLC) method applicable to the determination
of the compounds listed above in municipal and industrial discharges. When this method is used
to analyze unfamiliar samples for any or all of the compounds above, compound identification
should be supported by at least one additional qualitative technique. This method describes
analytical conditions for a second HPLC column that can be used to confirm measurements made
with the primary column.
1 .4 This method is restricted to use by or under the supervision of analysts experienced in the
operation of liquid chromatographs and in the interpretation of liquid chromatograms.
2. SUMMARY OF METHOD
2.1 The carbamate/amide pesticides are removed from the sample matrix by extraction with methylene
chloride. The extract is dried, exchanged to HPLC mobile phase and analyzed by liquid
chromatography with ultraviolet (IJV) detection.
3. INTERFERENCES
3.1 Solvent, reagents, glassware, and other sample processing hardware may yield discrete artifacts
and/or elevated baselines causing misinterpretation of liquid chromatograms. All of these
materials must be demonstrated to be free from interferences under the conditions of the analysis
by running laboratory reagent blanks as described in Section 9.1.
367
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Method 632.1
3.1.1 The use of high-purity reagents and solvents helps to minimize interference problems.
Purification of solvents by distillation in all-glass systems may be required.
3.1.2 Glassware must be scrupulously cleaned 1 . Clean all glassware as soon as possible after
use by rinsing with the last solvent used in it. This should be followed by detergent
washing with hot water and rinses with. tap water and reagent water. It should then be
drained dry and heated in a muffle furnace at 400°C for 15 to 30 minutes. Solvent rinses
with acetone and pesticide-quality hexane may be substituted for the heating. Volumetric
ware should not be heated in a muffle furnace. After drying and cooling, glassware
should be sealed and stored in a clean environment to prevent any accumulation of dust
or other contaminants. Store the glassware inverted or capped with aluminum foil.
3.2 Matrix interferences may be caused by UV-active contaminants that are coextracted from the
samples. The extent of matrix interferences will vary considerably from source to source,
depending upon the nature and diversity of the industrial complex or municipality being sampled.
Unique samples may require cleanup approaches to achieve the detection limits listed in Table 1.
4. SAFETY
4.1 The toxicity or carcinogenicity of each reagent used in this method has not been precisely defined;
however, each chemical compound should be treated as a potential health hazard. From this
viewpoint, exposure to these chemicals must be reduced to the lowest possible level by whatever
means available. The laboratory is responsible for maintaining a current awareness file of OSHA
regulations regarding the safe handling of the chemicals specified in this method. A reference file
of material data handling sheets should also be made available to all personnel involved in the
chemical analysis. Additional references to laboratory safety are available and have been
identified 2 for the information of the analyst.
5. APPARA TUS AND EQUIPMENT
5.1 Sample Containers: Narrow-mouth glass bottles, 1-L or 1-quart volume, equipped with
polytetrafluoroethylene (PTFE)-lined screw-caps. Wide-mouth glass bottles, 1-quart volume,
equipped with PTFE-lined screw-caps may also be used. Prior to use, wash bottles and cap liners
with detergent and rinse with tap and distilled water. Allow the bottles and cap liners to air dry,
then muffle the bottles at 400°C for 1 hour. After cooling, rinse the bottle and cap liners with
hexane, seal the bottles, and store in a dust-free environment.
5.1.1 Automatic sampler (optional): Must incorporate glass sample containers for the
collection of a minimum of 250 mL. Sample containers must be kept refrigerated at 4°C
and protected from light during compositing. If the sampler uses a peristaltic pump, a
minimum length of compressible silicone rubber tubing may be used. Before use,
however, the compressible tubing should be thoroughly rinsed with methanol, followed
by repeated rinsings with reagent water to minimize the potential for contamination of
the sample. An integrating flow meter is required to collect flow-proportional
composites.
5.2 Rotary evaporator: With 24/40 joints and associated water bath and vacuum for operation at
reduced pressure (Servo Instruments VE-1000-B or equivalent).
368
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Method 632.1
5.3 High performance liquid chromatography (HPLC) Apparatus: Analytical system complete with
liquid chromatograph and all required accessories including syringes, analytical columns, and
mobile phases. The system must be compatible with the specified detectors and strip-chart
recorder. A data system is recommended for measuring peak areas.
5.3.1 Gradient pumping system.
5.3.2 Injector valve (Rheodyne 7125 or equivalent) with 100- .&L loop.
5.3.3 Column 1: 250 mm long by 4.0 mm ID, stainless steel, packed with reverse-phase
Ultrasphere ODS, 5 , or equivalent.
5.3.4 Column 2: 250 nun long by 4.6 mm ID, packed with reverse phase Dupont Zorbax
ODS, 10 JL, or equivalent.
5.3.5 Ultraviolet detector, variable wavelength, capable of monitoring at 254 nm.
5.3.6 Strip-chart recorder compatible with detector, 250-mm. (A data system for measuring
peak areas is recommended.)
5.4 Boiling flask: 250-mL, flat-bottom, 24/40 joint.
5.5 Drying column: Approximately 400 mm long by 20 mm ID borosilicate glass, equipped with
coarse-fitted bottom plate.
5.6 Miscellaneous.
5.6.1 Balance: Analytical, capable of accurately weighing to the nearest 0.0001 g.
5.6.2 Separatory funnels: 2-L, equipped with PTFE stopcocks.
5.6.3 Boiling chips: Approximately 10/40 mesh. Heat to 400°C for 30 minutes or perform
a Soxhiet extraction with methylene chloride for 2 hours.
5.6.4 Standard solution storage containers: 15-mL bottles with PTFE-lined screw-caps.
5.6.5 Volumetric flasks: 5- and 10-mL,Class A.
5.6.6 Pasteur pipettes with bulbs.
6. REA GENTS AND CONSUMABLE MA TEPJALS
6.1 Reagents.
6.1.1 Acetone, acetonitrile, hexane, and methylene chloride: Demonstrated to be free of
analytes and interferences.
6.1.2 Reagent water: Reagent water is defined as a water in which an interferent is not
observed at the method detection limit of each parameter of interest.
6.1.3 Sodium sulfate: Granular, anhydrous. Condition by heating at 400°C for 4 hours in a
shallow tray.
6.1.4 HPLC Mobile Phase, Column 1: Add 400 mL of acetonitrile to a 1-L volumetric flask
and dilute to volume with reagent water.
6.1.5 HPLC Mobile Phase, Column 2: Add 550 mL of acetonitrile to a 1-L volumetric flask
and dilute to volume with reagent water.
6.1.6 Sodium hydroxide solution (1.ON): Dissolve 40 g of NaOH in reagent water and dilute
to 1,000 mL.
369
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Method 632.1
6.1.7 Sodium chloride: ACS, crystals.
6.1.8 Sodium thiosulfate: ACS, granular.
6.1.9 Sulfuric acid solution (1 + 1): Slowly add 50 mL of H 2 S0 4 (specific gravity 1.84) to
50 mL of reagent water.
6.2 Standard stock solutions (1.00 &g/ iL): These solutions may be purchased as certified solutions
or prepared from pure standard materials using the following procedures.
6.2.1 Prepare stock standard solutions by accurately weighing about 0.0100 g of pure material.
Dissolvethe material in pesticide-quality (9:1) acetonitrile/acetone and dilute to volume
in a 10-mL volumetric flask. Larger volumes can be used at the convenience of the
analyst. When compound purity is certified at 96% or greater, the weight can be used
without correction to calculate the concentration of the stock standard. Commercially
prepared stock standards can be used at any concentration if they are certified by the
manufacturer or by an independent source.
6.2.2 Transfer the stock standards to FFFE-sealed screw-cap bottles. Store at 4°C and protect
from light. Stock standards should be checked frequently for signs of degradation or
evaporation, especially just prior to preparing calibration standards from them.
6.2.3 Stock standards must be replaced after 6 months, or when comparison with quality
control check samples indicates a problem.
7. SAMPLE COLLECTiON. PRESERVATiON, AND STORAGE
7.1 Collect all samples in duplicate. Grab samples must be collected in glass containers.
Conventional sampling practices 5 should be followed, except that the bottle must not be prewashed
with sample before collection.
7.2 The samples must be iced or refrigerated at 4°C from the time of collection until extraction.
Chemical preservatives should not be used in the field unless more than 24 hours will elapse
before delivery to the laboratory. If the samples will not be extracted within 48 hours of
collection, the sample should be adjusted to a pH of 2.0 to 4.0 with sulfuric acid, and add 35 mg
of sodium thiosulfate per liter of sample for each part per million of free chlorine.
7.3 All samples must be extracted within 7 days and completely analyzed within 30 days of extraction.
8. CALIBRATiON
8.1 Establish liquid chromatographic operating parameters equivalent to those indicated in Table 1.
The chromatographic system can be calibrated using the external st2ndard technique (Section 8.2).
8.2 External standard calibration procedure.
8.2.1 Prepare calibration standards at a minimum of three concentration levels of the analytes
by adding volumes of the stock standard to a volumetric flask and diluting to volume with
HPLC mobile phase. One of the standards should be at a concentration near, but greater
than, the EDL, and the other concentrations should correspond to the expected range of
concentrations found in real samples or should define the working range of the detector.
8.2.2 Using injections of 100 L of each calibration standard, tabulate peak height or area
response against the mass injected. The results are used to prepare a calibration curve
370
-------
Method 632.1
for the analytes. Alternatively, if the ratio of response to amount injected (calibration
factor) is a constant over the working range (<10% relative standard deviation, RSD),
linearity of the calibration curve can be assumed and the average ratio or calibration
factor can be used in place of a calibration curve.
8.2.3 The working calibration curve or calibration factor must be verified on each working day
by the measurement of one or more calibration standards. If the response for any analyte
varies from the predicted response by more than ± 10%, the test must be repeated using
a fresh calibration standard. Alternatively, a new calibration curve or factor must be
prepared.
9. QUALITY CONTROL
9.1 Monitoring for interferences.
9.1 .1 Analyze a laboratory reagent blank each time a set of samples is extracted. A laboratory
reagent blank is an aliquot of reagent water. If the reagent blank contains a reportable
level of the analytes, immediately check the entire analytical system to locate and correct
for possible interferences and repeat the test.
9.2 Assessing accuracy.
9.2.1 After every ten samples, and preferably in the middle of each day, analyze a laboratory
control standard. Calibration standards may not be used for accuracy assessments and
the laboratory control standard may not be used for calibration of the analytical system.
9 .2.1.1 Laboratory control standard concentrate: From stock standards prepared as
described in Section 6.2, prepare a laboratory control standard concentrate
that contains the analytes at a concentration of 10 j g/mL in acetonitrile. 6
9.2.1.2 Laboratory control standard: Using a pipette, add 1.0 mL of the laboratory
control standard concentrate to a 1-L aliquot of reagent water.
9.2.1.3 Analyze the laboratory control standard as described in Section 10. Calculate
the percent recovery (Pj with the equation:
Equation 1
100s.
• = i
where
S. = The analytical results from the laboratory control standard, in pg/L
= The known concentration of the spike, in gIL
9.2.2 At least annually, the laboratory should participate in formal performance evaluation
studies, where solutions of unknown concentrations are analyzed and the performance of
all participants is compared.
371
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Method 632.1
9.3 Assessing precision.
9.3.1 Precision assessments for this method are based upon the analysis of field duplicates
(Section 7.1). Analyze both aliquots for at least 10% of all samples. To the extent
practical, the samples for duplication should contain reportable levels of the analytes.
9.3.2 Calculate the relative range 6 (RRJ with the equation:
Equation 2
100R
RR
;;
where
= The ab volWe difference between the duplicate measurements X and X , in g/L
x÷;
The a rage , ation jbwzd 2 in pg/L
9.3.3 Individual relative range measurements are pooled to determine average relative range
or to develop an expression of relative range as a function of concentration.
10. PROCEDURE
10.1 Sample extraction.
10.1.1 Mark the water meniscus on the side of the sample bottle for later determination of
sample volume. Pour the entire sample into a 2-L separatory funnel. Check the pH of
the sample with wide-range pH paper and adjust to within the range of 6.5 to 7.5 with
sodium hydroxide or sulfuric acid by slow addition and thorough mixing. Add 200 g of
sodium chloride, and mix to dissolve.
10.1.2 Add 60 inL of methylene chloride to the sample bottle and shake for 30 seconds to rinse
the walls. Transfer the solvent to the separatory funnel and extract the sample by shaking
the funnel for 2 minutes with periodic venting to release vapor pressure. Allow the
organic layer to separate from the water phase for a minimum of 10 minutes. If the
emulsion interface between layers is more than one-third the volume of the solvent layer,
the analyst must employ mechanical techniques to complete the phase separation. The
optimum technique depends on the sample, but may include stirring, filtration of the
emulsion through glass wool, or centrifugation. Collect the extract in a 250 -mL
Erlenmeyer flask.
10.1.3 Add an additional 60-mL volume of methylene chloride to the sample bottle and complete
the extraction procedure a second time, combining the extracts in the Erlenmeyer flask.
10.1.4 Perform a third extraction in the same manner. Pour the combined extract through a
drying column containing about 10 cm of anhydrous sodium sulfate, collecting the
extract in a 250-mL flat-bottom boiling flask. Rinse the Erlenmeyer flask and column
with about 30 mL of methylene chloride to complete the transfer.
372
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Method 632.1
10.1.5 Concentrate the combined methylene chloride extracts to about 1 mL on a rotary
evaporator with bath temperature between 35 and 40°C. Add 15 mL of acetonitrile, and
reconcentrate to about 1 mL. Transfer the extract to a 1O-mL volumetric flask. Rinse
the boiling flask with about 1 mL of acetonitrile, and transfer to the volumetric flask.
A 5-mL syringe is recommended for this operation. Rinse the boiling flask further with
a 1-mL portion of acetonitrile, and transfer to the volumetric flask.
10.1.6 Add exactly 5.0 mL of HPLC-grade water to the flask, and dilute to 10 mL with
acetonitrile. If the extracts will be stored longer than 2 days, they should be transferred
to PTFE-sealed screw-cap bottles. If the sample extract requires no cleanup, proceed
with chromatographic analysis. If the sample requires cleanup, proceed to Section 10.2.
10.1.7 Determine the original sample volume by refilling the sample bottle to the mark and
transferring the liquid to a 1,000-mL graduated cylinder. Record the sample volume to
the nearest 5 mL.
10.2 Cleanup and separation.
10.2.1 Cleanup procedures may not be necessary for a relatively clean sample matrix. If
particular circumstances demand the use of a cleanup procedure, the analyst must
determine the elution profile and demonstrate that the recovery of each compound of
interest is no less than 85%.
10.2.2 Proceed with liquid chromatography as described in Section 10.3.
10.3 Liquid chromatography analysis.
10.3.1 Table 1 summarizes the recommended operating conditions for the liquid chromatograph.
Included in this table are the estimated retention times and estimated detection limits that
can be achieved by this method. An example of the separation achieved by the primary
column of the analytes is shown in Figures 1 and 2. Other columns, chromatographic
conditions, or detectors may be used if data quality comparable to Table 2 is achieved.
10.3.2 Calibrate the system daily as described in Section 8.
10.3.3 Inject 100 L of the sample extract. Monitor the column eluent at 254 nm. Record the
resulting peak size in area or peak height units.
10.3.4 The retention-time window used to make identifications should be based upon
measurements of actual retention-time variations of standards over the course of a day.
Three times the standard deviation of a retention time for a compound can be used to
calculate a suggested window size; however, the experience of the analyst should weigh
heavily in the interpretation of chromatograms.
10.3.5 If the response for the peak exceeds the working range of the system, dilute the sample
with mobile phase and reanalyze.
10.3.6 If the measurement of the peak response is prevented by the presence of interferences,
cleanup is required.
11. CALCULATIONS
11.1 Determine the concentration of analytes in the sample.
373
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Method 632.1
11.1.1 Calculate the amount of analytes injected from the peak response using the calibration
curve or calibration factor in Section 8.2.2. The concentration in the sample can be
calculated from the equation:
Equation 3
( A)(V )
Concentration, 1 ig/L = _____
(V)(V)
where
A = Amount of material injected, in ng
V, = Voiwne of extract injected, in 1 uL
V = Volume of total extract, in jiL
V = Volume of water extracted, in niL
11.2 Report results in milligrams per liter without correction for recovery data. When duplicate and
spiked samples are analyzed, report all data obtained with the sample results.
12. METHOD PERFORMANCE
12.1 The EDLc and associated chromatographic conditions for the analytes are listed in Table i. The
EDL is defined as the minimum response being equal to 5 times the background noise, assuming
a l0-mL final extract volume of a 1-L sample and an HPLC injection volume of 100 jiL.
12.2 Single-operator accuracy and precision studies were conducted by Environmental Science and
Engineering, Inc., in the designated matrix. The results of these studies are presented in Table 2.
374
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Method 632.1
References
1. ASTM Annual Book of Standards, Part 31, D3694, “Standard Practice for Preparation of Sample
Containers and for Preservation,” American Society for Testing and Materials, Philadelphia,
Pennsylvania, p. 679, 1986.
2. “Carcinogens - Working with Carcinogens,” Department of Health, Education, and Welfare,
Public Health Service, Center for Disease Control, National Institute for Occupational Safety and
Health, Publication No. 77-206, Aug. 1977.
3. “OSHA Safety and Health Standards, General Industry” (29 CFR 1910), Occupational Safety and
Health Administration, OSHA 2206 (Revised, January 1976).
4. “Safety in Academic Chemistry Laboratories,” American Chemical Society Publication,
Committee on Chemical Safety, 3rd Edition, 1979.
5. ASTM Annual Book of Standards, Part 31, D3370, “Standard Practice for Sampling Water,”
American Society for Testing and Materials, Philadelphia, Pennsylvania, p. 76, 1986.
6. “Handbook for Analytical Quality Control in Water and Wastewater Laboratories,”
EPA-600/4-79-019, U.S. Environmental Protection Agency, Environmental Monitoring, and
Support Laboratory, Cincinnati, Ohio, March 1979.
7. “Evaluation of Ten Pesticides,” U.S. Environmental Protection Agency, Contract 68-03-1760,
Task No. 11, U.S. Environmental Protection Agency, Environmental Monitoring and Support
Laboratory, Cincinnati, Ohio (In preparation.).
375
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Method 632.1
Table 1. Chromatographic Conditions and Estimated Detection Limits
Parameter Retention Time (Minutes)
Estimated Detection
Column 1 Column 2 Limit (,ig/L)
Vacor (RH 787) 6.0 3.8 0.20
Propanil 12.4 6.9 0.85
Napropamide 15.2 9.5 0.31
Column 1: 25 cm long by 4 mm ID, stainless steel, packed with Ultrasphere ODS (particle size 5 );
mobile phase: 40% acetonitrilelHPLC water programmed to 65% acetonitrile/HPLC water over 10
minutes at a flow rate of 1.0 mL/min at ambient temperature.
Column 2: 25 cm long by 4.6 mm ID, stainless steel, packed with Zorbax ODS (DuPont); mobile phase:
Isocratic elution with 55% acetonitrilelHPLC water at a flow rate of 1.0 mL/min for 6 minutes then
linear flow gradient to 1.5 mL/min over 3 minutes at ambient temperature.
Table 2. Single-Laboratory Accuracy and Precision
Spike Number Average Standard
Matdx Range of Percent Deviation
Parameter Type* (pg/L) RepLicates Recovery (%)
Napropamide 1 11.5 7 113.8 15.7
1 597.0 7 104.0 16.0
Propanil 1 14.0 7 99.8 12.4
1 676.0 7 96.4 7.6
Vacor (R11787) 1 12.9 7 98.2 17.5
1 655.0 7 111.2 5.2
* 1 = Spiked municipal wastewater
376
-------
Method 632.1
Retention Time (minutes)
, Napropamide
A5200264
HPLC Chromatogram of Carbamates/AmideS on Column 1.
Vacor
/
Carbaryl
/
0
6.0
8.0 10.0 12.0 14.0 16.0
Figure 1.
377
-------
? kthod 6 1
Vacor
/ Carbaryl
Propanit
/
Napropamide
0 2.0 4.0 6.0 8.0 10.0 12.0 14.0 16.0
Ratemion Tkne (minutes)
Figure 2. HPLC Chromatograrn of Carbamate/Amides in
Wastewater Extract on CoLumn 1.
378
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Method 633
The Determination of
Organonitrogen Pesticides in
Municipal and Industrial
Waste water
-------
Method 633
The Determination of Organonitrogen Pesticides in Municipal and
Industrial Waste water
SCOPE AND APPLICA TION
1.1 This method covers the determination of certain organonitrogen pesticides. The following
parameters can be determined by this method:
Parameter STORET No. CAS No.
Bromacil 314-40-9
Deet 134-62-3
Hex azinone 51235-04-2
Metribuzin 81408 21087-64-9
Terbacil 5902-51-2
Triadimefon 43121-43-3
Tricyclazole 41814-78-2
1.2 This is a gas chromatographic (GC) method applicable to the determination of the compounds
listed above in industrial and municipal discharges as provided under 40 CFR 136.1. Any
modification of this method beyond those expressly permitted shall be considered a major
modification subject to application and approval of alternative test procedures under 40 CFR 136.4
and 136.5.
1.3 The method detection limit (MDL, defined in Section 15) for five of the parameters are listed in
Table 1. The MDL for a specific wastewater may differ from those listed, depending upon the
nature of interferences in the sample matrix.
1 .4 This method is restricted to use by or under the supervision of analysts experienced in the use of
gas chromatography and in the interpretation of gas chromatograms. Each analyst must
demonstrate the ability to generate acceptable results with this method using the procedure
described in Section 8.2.
1.5 When this method is used to analyze unfamiliar samples for any or all of the compounds above,
compound identifications should be supported by at least one additional qualitative technique.
Section 14 provides gas chromatograph/rnass spectrometer (GCIMS) criteria appropriate for the
qualitative confirmation of compound identifications.
2. SUMMARY OF METHOD
2.1 A measured volume of sample, approximately 1 L, is extracted with methylene chloride using a
separatory funnel. The methylene chloride extract is dried and exchanged to acetone during
381
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Method 633
concentration to a volume of 10 mL or less. Gas chromatographic conditions are described which
permit the separation and measurement of the compounds in the extract by gas chromatography
with a therm ionic bead detector.’
3. iNTERFERENCES
3.1 Method interferences may be caused by contaminants in solvents, reagents, glassware, and other
sample-processing apparatus that lead to discrete artifacts or elevated baselines in gas
chromatograms. All reagents and apparatus must be routinely demonstrated to be free from
interferences under the conditions of the analysis by running laboratory reagent blanks as
described in Section 8.5.
3.1.1 Glassware must be scrupulously cleaned. 2 Clean all glassware as soon as possible after
use by thoroughly rinsing with the last solvent used in it. Follow by washing with hot
water and detergent and thorough rinsing with tap and reagent water. Drain dry and heat
in an oven or muffle furnace at 400°C for 15 to 30 minutes. Do not heat volumetric
ware. Thermally stable materials, such as PCBs, may not be eliminated by this
treatment. Thorough rinsing with acetone and pesticide-quality hexane may be substituted
for the heating. After drying and cooling, seal and store glassware in a clean
environment to prevent any accumulation of dust or other contaminants. Store inverted
or capped with aluminum foil.
3.1.2 The use of high-purity reagents and solvents helps to minimize interference problems.
Purification of solvents by distillation in all-glass systems may be required.
3.2 Matrix iflterferences may be caused by contaminants that are coextracted from the sample. The
extent of matrix interferences will vary considerably from source to source, depending upon the
nature and diversity of the industrial complex or municipality sampled. Unique samples may
require special cleanup approaches to achieve the MDL listed in Table I.
4. SAFETY
4.1 The toxicity or carcinogenicity of each reagent used in this method has not been precisely defined;
however, each chemical compound must be treated as a potential health hazard. From this
viewpoint, exposure to these chemicals must be reduced to the lowest possible level by whatever
means available. The laboratory is responsible for maintaining a current awareness file of OSHA
regulations regardIng the safe handling of the chemicals specified in this method. A reference file
of material data handling sheets shouLd also be made available to all personnel involved in the
chemical analysis. Additional references to laboratory safety are available and have been
identified” for the information of the analyst.
5. APPARA TUS AND MA TERIALS
5.1 Sampling equipment, for discrete or composite sampling.
5.1.1 Grab-sample bottle: Amber borosiicate or flint glass, 1-L or 1-quart volume, fitted with
screw-caps lined with TFE-fluorocarbon. Aluminum foil may be substituted for TFE if
the sample is not corrosive. If amber bottles are not available, protect samples from
-------
Method 633
light. The container and cap liner must be washed, rinsed with acetone or methylene
chloride, and dried before use to minimize contamination.
5.1.2 Automatic sampler (optional): Must incorporate glass sample containers for the
collection of a minimum of 250 mL. Sample containers must be kept refrigerated at 4°C
and protected from light during compositing. If the sampler uses a peristaltic pump, a
minimum length of compressible silicone rubber tubing may be used. Before use,
however, the compressible tubing must be thoroughly rinsed with methanol, followed by
repeated rinsings with reagent water to minimize the potential for contamination of the
sample. An integrating flow meter is required to collect flow-proportional composites.
5.2 Glassware. (All specifications are suggested. Catalog numbers are included for illustration only.)
5.2.1 Separatory funnel: 2000-mL, with TFE-fluorocarbon stopcock, ground-glass or TFE
stopper.
5.2.2 Drying column: Chromatographic column 400 mm long by 19 mm ID with coarse-fitted
disc.
5.2.3 Concentrator tube, Kuderna-Danish: 10-mL, graduated (Kontes K-570050-1025 or
equivalent). Calibration must be checked at the volumes employed in the test. Ground-
glass stopper is used to prevent evaporation of extracts.
5.2.4 Evaporative flask, Kuderna-Danish: 500-mL (Kontes K-570001-0500 or equivalent).
Attach to concentrator tube with springs.
5.2.5 Snyder column, Kuderna-Danish: Three-ball macro (Kontes K-503000-0121 or
equivalent).
5.2.6 Vials: Amber glass, 10- to 15-mL capacity with TFE-fluorocarbon-lined screw-cap.
5.3 Boiling chips: Approximately 10/40 mesh. Heat at 400°C for 30 minutes or performed Soxhlet
extraction with methylene chloride.
5.4 Water bath: Heated, with concentric ring cover, capable of temperature control (±2°C). The
bath should be used in a hood.
5.5 Balance: Analytical, capable of accurately weighing to the nearest 0.0001 g.
5.6 Gas chromatograph: Analytical system complete with gas chromatograph suitable for on-column
injection and all required accessories including syringes, analytical columns, gases, detector, and
strip-chart recorder. A data system is recommended for measuring peak areas.
5.6.1 Column 1: 180cm long by 2 mm ID glass, packed with 3% SP-2250DB on Supelcoport
(100/120 mesh) or equivalent. Operation of this column at high temperatures will
seriously reduce its useful period of performance. This column was used to develop the
method performance statements in Section 15. Alternative columns may be used in
accordance with the provisions described in Section 12.1.
5.6.2 Column 2: 180 cm long by 2 mm ID glass, packed with 3% SP-2401 on Supelcoport
(100/120 mesh) or equivalent.
5.6.3 Detector: Thermionic bead in the nitrogen mode. This detector has proven effective in
the analysis of wastewaters for the parameters listed in the scope and was used to develop
the method performance statements in Section 15. Alternative detectors, including a mass
spectrometer, may be used in accordance with the provisions described in Section 12.1.
383
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Method 633
6. REAGENTS
6.1 Reagent water: Reagent water is defined as a water in which an interferent is not observed at the
method detection limit of each parameter of interest.
6.2 Acetone, methylene chloride: Pesticide-quality or equivalent.
6.3 Sodium sulfate: ACS, granular, anhydrous. Condition by heating in a shallow tray at 400°C for
a minimum of 4 hours to remove phthalates and other interfering organic substances.
Alternatively, heat 16 hours at 450 to 500°C in a shallow tray or perform a Soxhlet extraction
with methylene chloride for 48 hours.
6.4 Stock standard solutions (1.00 pg/ L): Stock standard solutions may be prepared from pure
standard materials or purchased as certified solutions.
6.4.1 Prepare stock standard solutions by accurately weighing approximately 0.0100 g of pure
material. Dissolve the material in pesticide-quality acetone and dilute to volume in a
10-mi volumetric flask. Larger volumes may be used at the convenience of the analyst.
If compound purity is certified at 96% or greater, the weight may be used without
correction to calculate the concentration of the stock standard. Commercially prepared
stock standards may be used at any concentration if they are certified by the manufacturer
or by an independent source.
6.4.2 Transfer the stock standard solutions into TFE-fiuorocarbon-sealed screw-cap vials.
Store at 4°C and protect from light. Frequently check stock standard solutions for signs
of degradation or evaporation, especially just prior to preparing calibration standards
from them.
6.4.3 Stock standard solutions must be replaced after 6 months, or sooner if comparison with
check standards indicates a problem.
7. CALIBRATION
7.1 Establish gas chromatographic operating parameters equivalent to those indicated in Table 1. The
gas chromatographic system may be calibrated using either the external standard technique
(Section 7.2) or the internal standard technique (Section 7.3).
7.2 External standard calibration procedure.
7.2.1
For each parameter of interest, prepare calibration standards at a minimum of three
concentration levels by adding accurately measured volumes of one or more stock
standards to a volumetric flask and diluting to volume with acetone. One of the external
standards should be representative of a concentration near, but above, the method
detection limit. The other concentrations should correspond to the range of
concentrations expected in the sample concentrates or should define the working range
of the detector.
7.2.2 Using injections of 1 to 5 L of each calibration standard, tabulate peak height or area
responses against the mass injected. The results can be used to prepare a calibration
curve for each parameter. Alternatively, the ratio of the response to the mass injected,
defined as the calibration factor (CF), may be calculated for each parameter at each
standard concentration. If the relative standard deviation of the calibration factor is less
384
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Method 633
than 10% over the working range, the average calibration factor can be used in place of
a calibration curve.
7.2.3 The working calibration curve or calibration factor must be verified on each working
shift by the measurement of one or more calibration standards. If the response for any
parameter varies from the predicted response by more than ± 10%, the test must be
repeated using a fresh calibration standard. Alternatively, a new calibration curve or
calibration factor must be prepared for that parameter.
7.3 Internal standard calibration procedure: To use this approach, the analyst must select one or more
internal standards similar in analytical behavior to the compounds of interest. The analyst must
further demonstrate that the measurement of the internal standard is not affected by method or
matrix interferences. Due to these limitations, no internal standard applicable to all samples can
be suggested.
7.3.1 Prepare calibration standards at a minimum of three concentration levels for each
parameter of interest by adding volumes of one or more stock standards to a volumetric
flask. To each calibration standard, add a known constant amount of one or more
internal standards, and dilute to volume with acetone. One of the standards should be
representative of a concentration near, but above, the method detection limit. The other
concentrations should correspond to the range of concentrations expected in the sample
concentrates, or should define the working range of the detector.
7.3.2 Using injections of 1 to 5 p L of each calibration standard, tabulate the peak height or
area responses against the concentration for each compound and internal standard.
Calculate response factors (RF) for each compound as follows:
Equation 1
( A,) ( Ca )
RF = ________
(ALI)(CS)
where
A, = Response for the parameter to be measured
A = Response for the internal standard
= Concentration of the internal standard, in 1 igIL
C, = Concentration of the parameter to be measured, in gIL
If the RF value over the working range is constant, less than 10% relative standard
deviation, the RF can be assumed to be invariant and the average RF may be used for
calculations. Alternatively, the results may be used to plot a calibration curve of
response ratios, A 1 /A against RF.
7.3.3 The working calibration curve or RF must be verified on each working shift by the
measurement of one or more calibration standards. If the response for any parameter
varies from the predicted response by more than ± 10%, the test must be repeated using
a fresh calibration standard. Alternatively, a new calibration curve must be prepared for
that compound.
385
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Method 633
7.4 Before using any cleanup procedure, the analyst must process a series of calibration standards
through the procedure to validate elution patterns and the absence of interference from the
reagents.
8. QUAUTY CONTROL
8.1 Each laboratory using this method is required to operate a formal quality control program. The
minimum requirements of this program consist of an initial demonstration of laboratory capability
and the analysis of spiked samples as a continuing check on performance. The laboratory is
required to maintain performance records to define the quality of data that is generated.
8.1.1 Before performing any analyses, the analyst must demonstrate the ability to generate
acceptable accuracy and precision with this method. This ability is established as
described in Section 8.2.
8.1.2 In recognition of the rapid advances occurring in chromatography, the analyst is
permitted certain options to improve the separations or lower the cost of measurements.
Each time such modifications to the method are made, the analyst is required to repeat
the procedure in Section 8.2.
8.1.3 The laboratory must spike and analyze a minimum of 10% of all samples to monitor
continuing laboratory performance. This procedure is described in Section 8.4.
8.2 To establish the ability to generate acceptable accuracy and precision, the analyst must perform
the following operations.
8.2.1 Select a representative spike concentration for each compound to be measured. Using
stock standards, prepare a quality control check sample concentrate in acetone, 1000
times more concentrated than the selected concentrations.
8.2.2 Using a pipette, add 1.00 mL of the check sample concentrate to each of a minimum of
four 1000-mL aliquots of reagent water. A representative wastewater may be used in
place of the reagent water, but one or more additional aliquots must be analyzed to
determine background levels, and the spike level must exceed twice the background level
lbr the test to be valid. Analyze the aliquots according to the method beginning in
Section 10.
8.2.3 Calculate the average percent recovery (R), and the standard deviation of the percent
recovery (s), for the results. Wastewater background corrections must be made before
R and s calculations are performed.
8.2.4 Using the appropriate data from Table 2, determine the recovery and single-operator
precision expected for the method, and compare these results to the values calculated in
Section 8.2.3. If the data are not comparable, review potential problem areas and repeat
the test.
8.3 The analyst must calculate method performance criteria and define the performance of the
laboratory for each spike concentration and parameter being measured.
8.3.1 Calculate upper and lower control limits for method performance as follows:
Upper Control Limit (UCL) = R + 3s
Lower Control Limit (LCL) = R - 3s
386
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Method 633
Where R and s are calculated as in Section 8.2.3. The UCL and LCL can be used to
construct control charts 6 that are useful in observing trends in performance.
8.3.2 The laboratory must develop and maintain separate accuracy statements of laboratory
performance for wastewater samples. An accuracy statement for the method is defined
as R ± s. The accuracy statement should be developed by the analysis of four aliquots
of wastewater as described in Section 8.2.2, followed by the calculation of R and s.
Alternatively, the analyst may use four wastewater data points gathered through the
requirement for continuing quality control in Section 8.4. The accuracy statements
should be updated regularly. 6
8.4 The laboratory is required to collect in duplicate a portion of their samples to monitor spike
recoveries. The frequency of spiked sample analysis must be at least 10% of all samples or one
spiked sample per month, whichever is greater. One aliquot of the sample must be spiked and
analyzed as described in Section 8.2. If the recovery for a particular parameter does not fall
within the control limits for method performance, the results reported for that parameter in all
samples processed as part of the same set must be qualified as described in Section 13.3. The
laboratory should monitor the frequency of data so qualified to ensure that it remains at or below
5%.
8.5 Before processing any samples, the analyst must demonstrate through the analysis of a 1-L aliquot
of reagent water that all glassware and teagents interferences are under control. Each time a set
of samples is extracted or there is a change in reagent, a laboratory reagent blank must be
processed as a safeguard against laboratory contamination.
8.6 It is recommended that the laboratory adopt additional quality assurance practices for use with this
method. •The specific practices that are most productive depend upon the needs of the laboratory
and the nature of the samples. Field duplicates may be analyzed to monitor the precision of the
sampling technique. When doubt exists over the identification of a peak on the chromatogram,
confirmatory techniques such as gas chromatography with a dissimilar column, specific element
detector, or mass spectrometer must be used. Whenever possible, the laboratory should perform
analysis of quality control materials and participate in relevant performance evaluation studies.
9. SAMPLE COLLECTiON, PRESERVATION, AND HANDliNG
9.1 Grab samples must be collected in glass containers. Conventional sampling practices 7 should be
followed; however, the bottle must not be prerinsed with sample before collection. Composite
samples should be collected in refrigerated glass containers in accordance with the requirements
of the program. Automatic sampling equipment must be as free as possible of plastic and other
potential sources of contamination.
9.2 The samples must be iced or refrigerated at 4°C from the time of collection until extraction.
9.3 All samples must be extracted within 7 days and completely analyzed within 40 days of extraction.
10. SAMPLE EXTRACTiON
10.1 Mark the water meniscus on the side of the sample bottle for later determination of sample
volume. Pour the entire sample into a 2-L separatory funnel.
387
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Method 633
10.2 Add 60 mL methylene chloride to the sample bottle, seal, and shake 30 seconds to rinse the inner
walls. Transfer the solvent to the separatory funnel and extract the sample by shaking the funnel
fur two minutes with periodic venting to release excess pressure. Allow the organic layer to
separate from the water phase for a minimum of 10 minutes. If the emulsion interface between
layers is more than one-third the volume of the solvent layer, the analyst must employ mechanical
techniques to complete the phase separation. The optimum technique depends upon the sample,
but may include stirring, filtration of the emulsion through glass wool, centrifugation, or other
physical methods. Collect the methylene chloride extract in a 250-mL Erlenmeyer flask.
10.3 Add a second 60-mL volume of methylene chloride to the sample bottle and repeat the extraction
procedure a second time, combining the extracts in the Erlenmeyer flask. Perform a third
extraction in the same manner.
10.4 Assemble a Kuderna-Danish (K-D) concentrator by attaching a 10-mL concentrator tube to a
500-mL evaporative flask. Other concentration devices or techniques may be used in place of the
K-D if the requirements of Section 8.2 are met.
10.5 Pour the combined extract through a drying column containing about 10 cm of anhydrous sodium
sulfate, and collect the extract in the K-D concentrator. Rinse the Erlenmeyer flask and column
with 20 to 30 mL of methylene chloride to complete the quantitative transfer.
10.6 Add one or two clean boiling chips to the evaporative flask and attach a three-ball Snyder column.
Prewet the Snyder column by adding about 1 mL methylene chloride to the top. Place the K-D
apparatus on a hot water bath, 60 to 65°C, so that the concentrator tube is partially immersed in
the hot water, and the entire lower rounded surface of the flask is bathed with hot vapor. Adjust
the vertical position of the apparatus and the water temperature as required to complete the
concentration in 15 to 20 minutes. At the proper rate of distillation, the balls of the column will
actively chatter but the chambers will not flood with condensed solvent. When the apparent
volume of liquid reaches I mL, remove the K-D apparatus and allow it to drain and cool for at
least 10 minutes.
10.7 Increase the temperature of the hot water bath to about 70°C. Momentarily remove the Snyder
column, add 50 mL of acetone and a new boiling chip and reauach the Snyder column. Pour
about I mL of acetone into the top of the Snyder column and concentrate the solvent extract as
before. Elapsed time of concentration should be 5 to 10 minutes. When the apparent volume of
liquid reaches I mL, remove the K-D apparatus and allow it to drain and cool for at least 10
minutes.
10.8 Remove the Snyder column and rinse the flask and its lower joint into the concentrator tube with
1 to 2 mL of hexane and adjust the volume to 10 mL. A 5-mL syringe is recommended for this
operation. Stopper the concentrator tube and store refrigerated if further processing will not be
performed immediately. If the extracts will be stored longer than 2 days, they should be
transferred to TFE-fluorocarbon-sealed screw-cap vials. Analyze by gas chromatography.
10.9 Determine the original sample volume by refilling the sample bottle to the mark and transferring
the water to a 1000-mi. graduated cylinder. Record the sample volume to the nearest 5 mL.
11. CLEANUP AND SEPARA TION
11.1 Cleanup procedures may not be necessary for a relatively clean sample matrix. If particular
circumstances demand the use of a cleanup procedure, the analyst must determine the elution
388
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Method 633
profile and demonstrate that the recovery of each compound of interest for the cleanup procedure
is no less than 85%.
12. GAS CHROMATOGRAPHY
12.1 Table 1 summarizes the recommended operating conditions for the gas chromatograph. Included
in this table are estimated retention times and method detection limits that can be achieved by this
method. An example of the separations achieved by Column 1 is shown in Figure 1. Other
packed columns, chromatographic conditions, or detectors may be used if the requirements of
Section 8.2 are met. Capillary (open-tubular) columns may also be used if the relative standard
deviations of responses for replicate injections are demonstrated to be less than 6% and the
requirements of Section 8.2 are met.
12.2 Calibrate the system daily as described in Section 7.
12.3 If the internal standard approach is being used, add the internal standard to sample extracts
immediately before injection into the instrument. Mix thoroughly.
12.4 Inject 1 to 5 1 LL of the sample extract using the solvent-flush technique. 8 Record the volume
injected to the nearest 0.05 L, and the resulting peak size in area or peak height units. An
automated system that consistently injects a constant volume of extract may also be used.
12.5 The width of the retention-time window used to make identifications should be based upon
measurements of actual retention-time variations of standards over the course of a day. Three
times the standard deviation of a retention time can be used to calculate a suggested window size
for a compound. However, the experience of the analyst should weigh heavily in the
interpretation of chromatograms.
12.6 If the response for the peak exceeds the working range of the system, dilute the extract and
reanalyze.
12.7 If the measurement of the peak response is prevented by the presence of interferences, cleanup
is required.
13. CALCULATiONS
13.1 Determine the concentration of individual compounds in the sample.
13.1.1 If the external standard calibration procedure is used, calculate the amount of material
injected from the peak response using the calibration curve or calibration factor in
Section 7.2.2. The concentration in the sample can be calculated as follows:
389
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Method 633
Equation 2
( A)(V )
Concentration, igIL = ______
(V 1 )(V)
where
A = Amount of material injected, in ng
V, = Volume of extract injected, in
V = Volume of total extract, in p.L
= Volume of water extracted, in mL
13.1.2 If the internal standard calibration procedure was used, calculate the concentration in the
sample using the response factor (RF) determined in Section 7.3.2 as follows:
Equation 3
Concentration, g/L = (A ,)(RF)(V 0 )
.
where
A, = Response for parameter to be measured
A , = Response for the internal standard
Amount of internal standard added to each extract,
V 0 = Volume of water extracted, in L
in
g
13.2 Report results in micrograms per liter without correction for recovery data. When duplicate and
spiked samples are analyzed, report all data obtained with the sample results.
13.3 For samples processed as part of a set where the laboratory spiked sample recovery falls outside
of the control limits in Section 8.3, data for the affected parameters must be labeled as suspect.
14. CC/MS CONFIRMA liON
14.1 It is recommended that GC/MS techniques be judiciously employed to support qualitative
compound identifications made with this method. The mass spectrometer should be capable of
scanning the mass range from 35 amu to a mass 50 amu above the molecular weight of the
compound. The instrument must be capable of scanning the mass range at a rate to produce at
least 5 scans per peak but not to exceed 7 seconds per scan utilizing a 70 V (nominal) electron
energy in the electron impact ionization mode. A GC to MS interface constructed of all glass or
glass-lined materials is recommended. A computer system should be interfaced to the mass
spectrometer that allows the continuous acquisition and storage on machine-readable media of all
mass spectra obtained throughout the duration of the chromatographic program.
14.2 Gas chromatographic columns and conditions should be selected for optimum separation and
performance. The conditions selected must be compatible with standard GC/MS operating
practices. Chromatographic tailing factors of less than 5.0 must be achieved. 9
390
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Method 633
14.3 At the beginning of each day that confirmatory analyses are to be performed, the GC/MS system
must be checked to see that all decafluorotriphenyl phosphine (DFFPP) performance criteria are
achieved.’ 0
14.4 To confirm an identification of a compound, the background-corrected mass spectrum of the
compound must be obtained from the sample extract and compared with a mass spectrum from
a stock or calibration standard analyzed under the same chromatographic conditions. It is
recommended that at least 25 ng of material be injected into the (iC/MS. The criteria below must
be met for qualitative confirmation.
14.4.1 All ions that are present above 10% relative abundance in the mass spectrum of the
standard must be present in the mass spectrum of the sample with agreement to ±10%.
For example, if the relative abundance of an ion is 30% in the mass spectrum of the
standard, the allowable limits for the relative abundance of that ion in the mass spectrum
for the sample would be 20 to 40%.
14.4.2 The retention time of the compound in the sample must be within 6 seconds of the same
compound in the standard solution.
14.4.3 Compounds that have very similar mass spectra can be explicitly identified by GCIMS
only on the basis of retention time data.
14.5 Where available, chemical ionization mass spectra may be employed to aid in the qualitative
identification process.
14.6 Should these MS procedures fail to provide satisfactory results, additional steps may be taken
before reanalysis. These may include the use of alternate packed or capillary GC columns or
additional cleanup (Section 11).
15. METHOD PERFORMANCE
15.1 The method detection limit (MDL) is defmed as the minimum concentration of a substance that
can be measured and reported with 99% confidence that the value is above zero.” The MDL
concentrations listed in Table 1 were obtained using reagent water. 1
15.2 In a single laboratory (‘West Cost Technical Services, Inc.), using effluents from pesticide
manufacturers and publicly owned treatment works (POTW), the average recoveries presented in
Table 2 were obtained.’ The standard deviations of the percent recoveries of these measurements
are also included in Table 2.
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Method 633
References
1. “Pesticide Methods Evaluation,” Letter Reports #6, 12A and 14 for EPA Contract No. 68-03-2697.
Available from U.S. Environmental Protection Agency, Environmental Monitoring and Support
Laboratory, Cincinnati, Ohio.
2. ASTM Annual Book of Standards, Part 31, D3694, “Standard Practice for Preparation of Sample
Containers and for Preservation, U American Society for Testing and Materials, Philadelphia,
Pennsylvania, p. 679, 1980.
3. “Carcinogens - Working with Carcinogens,” Department of Health, Education, and Welfare,
Public Health Service, Center for Disease Control, National Institute for Occupational Safety and
Health, Publication No. 77-206, Aug. 1977.
4. “OSHA Safety and Health Standards, General Industry,” (29 CFR 1910), Occupational Safety and
Health Administration, OSHA 2206, (Revised, January 1976).
5. “Safety in Academic Chemistry Laboratories,” American Chemical Society Publication,
Committee on Chemical Safety, 3rd Edition, 1979.
6. “Handbook for Analytical Quality Control in Water and Wastewater Laboratories,”
EPA-600/4-79-019, U. S. Environmental Protection Agency, Environmental Monitoring and
Support Laboratory - Cincinnati, Ohio, March 1979.
7. ASTM Annual Book of Standards, Part 31, D3370, “Standard Practice for Sampling Water,”
American Society for Testing and Materials, Philadelphia, Pennsylvania, p. 76, 1980.
8. Burke, LA., “Gas Chromatography for Pesticide Residue Analysis; Some Practical Aspects,”
Journal of the Association of Official Analytical Chemists, 48,1037 (1965).
9. McNair, H.M., and Bonelli, E.J., Basic Chromatography, Consolidated Printing, Berkeley,
California, p. 52,1969.
10. Eichelberger, J.W., Harris, L.E., and Budde, W.L. “Reference Compound to Calibrate Ion
Abundance Measurement in Gas Chromatography-Mass Spectroinetry,” Analytical Chemistiy, 47,
995 (1975).
11. Glaser, LA. et.al, “Trace Analysis for Wastewaters,” Environmental Science & Technology, 15,
1426 (1981).
392
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Method 633
Table 1. Chromatographic Conditions and Method Detection Limits
Method
Retention Detection
GC Time Limit
Parameter Column (Mm) (pg/LI
Terbacil la 2.1 ND
Bromacil la 3.7 2.38
Hexazinone la 7.6 0.72
Tricyclazole lb 3.5 ND
Metribuzin 2a 2.4 0.46
Triadimefon 2a 4.1 0.78
Deet 2b 4.6 3.39
ND = Not determined
Column la conditions: Supelcoport (100/120 mesh) coated with 3% SP-2250DB packed in a 180 cm long
by 2 mm ID glass column with nitrogen carrier gas at a flow rate of 30 mL/min. Column temperature,
programmed: initial 2 10°C, hold for 1 minute, then program at 10°C to 250°C and hold. A thermionic
bead detector in the nitrogen mode was used to calculate the MDL.
Column lb conditions: Same as Column la, except column temperature isothermal at 240°C.
Column 2a conditions: Supelcoport (100/120 mesh) coated with 3% SP-2401 packed in a 180 cm long
by 2 mm ID glass column with nitrogen carrier gas at a flow rate of 30 mL/min. Column temperature,
programmed: initial 160°C, programmed at injection at 10°C/mm to 230°C.
Column 2b conditions: Same as Column 2a, except temperature programmed: initial 130°C, hold for
1 minute, then program at 12°C/mm to 200°C.
393
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Method 633
Table 2. Single-Operator Accuracy and Precision
Number Mean Standard
Sample Spilce of Recovery Deviation
Parameter Type (pg/L) Repicates (%J (%)
Bromacil DW 5.0 7 92.2 13.9
MW 11.1 7 89.0 3.9
MW 333.0 7 95.0 0.8
Deet DW 5.8 7 99.1 18.4
MW 5.2 7 92.6 5.9
MW 515.0 7 94.2 2.2
Hexazinon DW 4.9 7 86.6 4.1
MW 10.1 7 92.2 5.3
MW 369.0 7 94.0 1.9
Metribuzin DW 5.2 6 98.2 2.7
Terbacil MW 32.8 7 106.7 3.6
MW 656.0 7 101.0 1.2
Triadmefon DW 5.2 6 126.0 6.0
PW 515.0 4 71.8 4.5
1W 154.5 7 70.4 3 8
Tricyclazole MW - 12.3 7 69.0 1.9
MW 303.0 7 98.0 1.2
DW= Reagent water
MW = Municipal wastewater
PW = Process water, pesticide manufacturing
1W = Industrial wastewater, pesticide manufacturing
394
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Method 633
Hexazinone
I I I I I I I I I I I I I I I I I
1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0
Retention Time (minutes)
Figure 1. Gas Chromatogram of Organonitrogen Pesticides on Column 1.
For Conditions, See Table 1.
Terbacil
Bromacil
0
395
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Method 633.1
The Determination of Neutral
Nitrogen- Containing Pes tic/des
in Municipal and Industrial
Waste waters
-------
Method 633.1
The Determination of Neutral Nitrogen-Containing Pesticides
in Municipal and Industrial Wastewaters
SCOPE AND APPLICATiON
1.1 This method covers the determination of certain neutral nitrogen containing pesticides. The
following parameters can be determined by this method:
Parameter CAS No.
Fenarimol 60168-88-9
MGK 264-A 113-48-4
MGK 264-B 113-48-4
MGK 326 136-45-8
Pronamide 23950-58-5
1.2 This is a gas chromatographic (GC) method applicable to the determination of the compounds
listed above in municipal and industrial discharges as provided under 40 CFR 136.1. Any
modification of this method beyond thàse expressly permitted shall be considered a major
modification subject to application and approval of alternative test procedures under 40 CFR 136.4
and 136.5.
1.3 The method detection limit (MDL, defined in Section 15) for each compound is listed in Table 2.
The MDL for a specific wastewater may differ from those listed, depending upon the nature of
interferences in the sample matrix.
1.4 The sample extraction and concentration steps in this method are similar to those in other 600-
series methods. Thus, a single sample may be extracted to measure the compounds included in
the scope of the methods. When cleanup is required, the concentration levels must be high
enough to permit selecting aliquots, as necessary, in order to apply appropriate cleanup
procedures.
1.5 This method is restricted to use by or under the supervision of analysts experienced in the use of
gas chromatography and in the interpretation of gas chromatograms. Each analyst must
demonstrate the ability to generate acceptable results with this method using the procedure
described in Section 8.2.
1.6 When this method is used to analyze unfamiliar samples for any or all of the compounds above,
compound identifications should be supported by at least one additional qualitative technique.
This method describes analytical conditions for a second gas chromatographic column that can be
used to confirm measurements made with the primary column. Section 14 provides gas
chroinatograph/mass spectrometer (GC/MS) criteria appropriate for the qualitative confirmation
of compound identifications.
399
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Method 633.1
2. SUMMARY OF METHOD
2.1 A measured volume of sample, approximately 1 L, is extracted with methylene chloride using a
separatory funnel. The methylene chloride extract is dried and concentrated to 1.0 mL. Gas
chromatographic conditions are described which permit the separation and measurement of the
compounds in the extract by alkali flame detector gas chromatography (GCIAFD). 1
2.2 This method provides an optional Florisil column cleanup procedure to aid in the elimination of
interferences which may be encountered.
3. INTERFERENCES
3.1 Method interferences may be caused by contaminants in solvents, reagents, glassware, and other
sample-processing apparatus that lead to discrete artifacts or elevated baselines in gas
chromatograms. All reagents and apparatus must be routinely demonstrated to be free from
interferences under the conditions of the analysis by running laboratory reagent blanks as
described in Section 8.5.
3.1.1
Glassware must be scrupulously cleaned. 2 Clean all glassware as soon as possible after
use by rinsing with the last solvent used in it. Follow by rinsing with hot water and
detergent and thorough rinsing with tap and reagent water. Drain dry, and heat in an
oven or muffle furnace at 400°C for 15 to 30 minutes. Do not heat volumetric ware.
Some thermally stable materials, such as PCBs, may not be eliminated by this treatment.
Thorough rinsing with acetone and pesticide-quality hexane may be substituted for the
heating. After drying and cooling, seal and store glassware in a clean environment to
prevent any accumulation of dust or other contaminants. Store inverted or capped with
aluminum foil.
3.1.2 The use of high-purity reagents and solvents helps to minimize interference problems.
Purification of solvents by distillation in all-glass systems may be required.
3.2 Matrix interferences may be caused by contaminants that are coextracted from the sample. The
extent of matrix interferences will vary considerably from source to source, depending upon the
nature and diversity of the industrial complex or municipality sampled. The cleanup procedure
in Section 11 can be used to overcome many of these interferences, but unique samples may
require additional cleanup approaches to achieve the MDL listed in Table 2.
4. SAFETY
4.1 The toxicity or carcinogenicity of each reagent used in this method has not been precisely defined;
however, each chemical compound should be treated as a potential health hazard. From this
viewpoint, exposure to these chemicals must be reduced to the lowest possible level by whatever
means available. The laboratory is responsible for maintaining a current awareness file of OSHA
regulations regarding the safe handling of the chemicals specified in this method. A reference file
of materials data handling sheets should also be made available to all personnel involved in the
chemical analysis. Additional references to laboratory safety are available and have been
identified 5 for the information of the analyst.
400
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Method 633.1
5. APPARA TUS AND MA TER1ALS
5.1 Sampling equipment, for discrete or composite sampling.
5.1.1 Grab-sample bottle: Amber borosilicate or flint glass, 1-L or 1-quart volume, fitted with
screw-caps lined with PTFE. Foil may be substituted for PTFE if the sample is not
corrosive. If amber bottles are not available, protect samples from light. The container
and cap liner must be washed, rinsed with acetone or methylene chloride, and dried
before use to minimize contamination.
5.1.2 Automatic sampler (optional): Must incorporate glass sample containers for the collection
of a minimum of 250 mL. Sample containers must be kept refrigerated at 4°C and
protected from light during compositing. If the sampler uses a peristaltic pump, a
minimum length of compressible silicone rubber tubing may be used. Before use,
however, the compressible tubing should be thoroughly rinsed with methanol, followed
by repeated rinsings with distilled water to minimize the potential for contamination of
the sample. An integrating flow meter is required to collect flow-proportional
composites.
5.2 Glassware. (All specifications are suggested. Catalog numbers are included for illustration only.)
5.2.1 Separatory funnel: 2000-mL, with PTFE stopcock.
5.2.2 Drying column: Chromatographic column 400 mm long by 10 mm ID with coarse fit.
5.2.3 Chromatographic column: 400 mm long by 19 mm ID with 250-mL reservoir at the top
and PTFE stopcock (Kontes K-420290 or equivalent).
5.2.4 Concentrator tube, Kuderna-Danish: 10-mL, graduated (Kontes K-570050-1025 or
equivalent). Calibration must be checked at the volumes employed in the test. A
ground-glass stopper is used to prevent evaporation of extracts.
5.2.5 Evaporative flask, Kuderna-Danish 500-mL (Kontes K-570001-0500 or equivalent).
Attach to concentrator tube with springs.
5.2.6 Snyder column, Kuderna-Danish: Three-ball macro (Kontes K-503000-0121 or
equivalent).
5.2.7 Snyder column, Kuderna-Danish: Two-ball micro (Kontes K-569001-0219 or equivalent).
5.2.8 Vials: Amber glass, 10- to 15-mL capacity with P’FFE-lined screw-cap.
5.2.9 Erlenmeyer flask: 250-mL.
5.2.10 Graduated cylinder: 1000-mL.
5.2.11 Beaker: 250-mL.
5.3 Boiling chips: Approximately 10/40 mesh carborundum. Heat to 400°C for 4 hours or perform
a Soxhlet extraction with methylene chloride.
5.4 Water bath: Heated, capable of temperature control (± 20°C). The bath should be used in a hood.
5.5 Balance: Analytical, capable of accurately weighing to the nearest 0.0001 g.
5.6 Gas chromatograph: Analytical system complete with gas chromatograph suitable for on-column
injection and all required accessories including syringes, analytical columns, gases, detector, and
strip-chart recorder. A data system is recommended for measuring peak areas.
401
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Method 633.1
5.6.1 Column 1: 180 cm long by 2 mm II) glass, packed with 3% SP-2250 on Supelcoport
(100/120 mesh) or equivalent. This column was used to develop the method performance
statements in Section 15. Alternative columns may be used in accordance with the
provisions described in Section 12.1.
5.6.2 Column 2: 180 cm long by 2 mm ID glass, packed with 3% SP-2100 on Supelcoport
(100/120 mesh) or equivalent.
5.6.3 Detector: Alkali flame detector (AFD), sometimes referred to as a nitrogen-phosphorous
detector (NPD) or a thermionic-specific detector (I’SD). This detector has proven
effective in the analysis of wastewaters for the compounds listed in the scope and was
used to develop the method performance statements in Section 15.
6. REAGENTS
6.1 Reagent water: Reagent water is defined as a water in which an interferent is not observed at the
method detection limit of each parameter of interest.
6.2 Methylene chloride, methanol, petroleum ether, ethyl ether, acetone: Distilled-in-glass quality or
equivalent. Ethyl ether must be free of peroxides as indicated by EM Quant test strips (available
from Scientific Products Co., Catalog No. P1126-8 and other suppliers). Procedures
recommended for removal of pes xides are provided with the test strips.
6.3 6N sodium hydroxide: Dissolve 24.0 g NaOH in 100 mL of reagent water.
6.4 6N sulfuric acid: Slowly add 16.7 mL of concentrated 11 2 S0 4 (94%) to about 50 mL of reagent
water . Dilute to 100 mL with reagent water.
6.5 Sodium sulfate: (ACS), granular, anhydrous; heated in a muffle furnace at 400°C overnight.
6.6 Florisil: PR grade (60/100 mesh). Purchase activated at 675°C and store in brown glass bottle.
To prepare for use, place 150 & in a wide-mouth jar and heat overnight at 160 to 170°C. Seal
tightly with Ffl E or aluminum-foil-lined screw-cap and cool to room temperature.
6.7 Stock standard solutions (1.00 &g/pL): Stock standard solutions can be prepared from pure
standard materials or purchased as certified solutions.
6.7.1 Prepare stock standard solutions by accurately weighing about 0.0100 g of pure material.
Dissolve the material in distilled-in-glass quality methanol and dilute to volume in a
10-mL volumetric flask. Larger volumes can be used at the convenience of the analyst.
If compound purity is certified at 96% or greater, the weight can be used without
correction to calculate the concentration of the stock standard. Commercially prepared
stock standards can be used at any concentration if they are certified by the manufacturer
or by an independent source.
6.7.2 Transfer the stock standard solutions into Ffl E-sea1ed screw-cap bottles. Store at 4°C
and protect from light. Frequently check stock standard solutions for signs of
degradation or evaporation, especially just prior to preparing calibration standards from
them.
6.7.3 Stock standard solutions must be replaced after 6 months, or sooner if comparison with
check standards indicates a problem.
402
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Method 633.1
7. CALIBRATION
7.1 Establish gas chromatographic operating parameters equivalent to those indicated in Table 2. The
gas chromatographic system can be calibrated using the external standard technique (Section 7.2)
or the internal standard technique (Section 7.3).
7.2 External standard calibration procedure.
7.2.1 For each compound of interest, prepare calibration standards at a minimum of three
concentration levels by adding volumes of one or more stock standards to a volumetric
flask and diluting to volume with acetone. One of the external standards should be at a
concentration near, but above, the method detection limit. The other concentrations
should correspond to the range of concentrations expected in the sample concentrates or
should define the working range of the detector.
7.2.2 Using injections of 1 to 5 L of each calibration standard, tabulate peak height or area
responses against the mass injected. The results can be used to prepare a calibration
curve for each compound. Alternatively, the ratio of the response to the mass injected,
defined as the calibration factor (CF), can be calculated for each compound at each
standard concentration. If the relative standard deviation of the calibration factor is less
than 10% over the working range, the average calibration factor can be used in place of
a calibration curve.
7.2.3 The working calibration curve or calibration factor must be verified on each working
shift by the measurement of one or more calibration standards. If the response for any
compound varies from the predicted response by more than ±10%, the test must be
repeated using a fresh calibration standard. Alternatively, a new calibration curve or
calibration factor must be prepared for that compound.
7.3 Internal standard calibration procedure: To use this approach, the analyst must select one or more
internal standards similar in analytical behavior to the compounds of interest. The analyst must
further demonstrate that the measurement of the internal standard is not affected by method or
matrix interferences. Due to these limitations, no internal standard applicable to all samples can
be suggested.
7.3.1 Prepare calibration standards at a minimum of three concentration levels for each
compound of interest by adding volumes of one or more stock standards to a volumetric
flask. To each calibration standard, add a known constant amount of one or more
internal standards, and dilute to volume with acetone. One of the standards should be
at a concentration near, but above, the method detection limit. The other concentrations
should correspond to the range of concentrations expected in the sample concentrates, or
should define the working range of the detector.
7.3.2 Using injections of 1 to 5 L of each calibration standard, tabulate the peak height or
area responses against the concentration for each compound and internal standard.
Calculate response factors (RF) for each compound as follows:
403
-------
Method 633.1
Equation 1
.RF = ( A,)(C,, )
(A,,)(C,)
*ere
A, = Response for the parameter to be measured
4, = Response for the internal standard
C,, = Concentratlén of the internal standard, in ,igIL
C, = Concentration of the parameter to be measured, in igIL
If the RF value over the working range is constant, less than 10% relative standard
deviation, the RF can be assumed to be invariant and the average RF can be used for
calculations. Alternatively, the results can be used to plot a calibration curve of response
ratios, AJA,, against RF.
7.3.3 The working calibration curve or RF must be verified on each working shift by the
measurement of one or more calibration standards. If the response for any compound
varies from the predicted response by more than ± 10%, the test must be repeated using
a fresh calibration standard. Alternatively, a new calibration curve must be prepared for
that compound.
7.4 Before using any cleanup procedure, the analyst must process a series of calibration standards
through the procedure to validate elution patterns and the absence of interferences from the
reagents.
8. QuAliTY CONTROL
8.1 Each laboratory using this method is required to operate a formal quality control program. The
nainnuuni requirements of this program consist of an initial demonstration of laboratory capability
and the analysis of spiked samples as a continuing check on performance. The laboratory is
required to maintain performance records to define the quality of data that is generated.
8.1.1 Before performing any analyses, the analyst must demonstrate the ability to generate
acceptable accuracy and precision with this method. This ability is established as
described in Section 8.2.
8.1.2 In recognition of the rapid advances occurring in chromatography, the analyst is
permitted certain options to improve the separations or lower the cost of measurements.
Each time such modifications to the method are made, the analyst is required to repeat
the procedure in Section 8.2.
8.1.3 The laboratory must spike and analyze a minimum of 10% of all samples to monitor
continuing laboratory performance. This procedure is described in Section 8.4.
8.2 To establish the ability to generate acceptable accuracy and precision, the analyst must perform
the following operations.
8.2.1 Select a representative spike concentration for each compound to be measured. Using
stock standards, prepare a quality control check sample concentrate in methanol, 1000
times more concentrated than the selected concentrations.
40
-------
Method 633.1
8.2.2 Using a pipette, add 1.00 mL of the check sample concentrate to each of a minimum of
four 1000-mL aliquots of reagent water. A representative wastewater may be used in
place of the reagent water, but one or more additional aliquots must be analyzed to
determine background levels, and the spike level must exceed twice the background level
for the test to be valid. Analyze the aliquots according to the method beginning in
Section 10.
8.2.3 Calculate the average percent recovery (R), and the standard deviation of the percent
recovery (s), for the results. Wastewater background corrections must be made before
R and s calculations are performed.
8.2.4 Using the appropriate data from Table 3, determine the recovery and single-operator
precision expected for the method, and compare these results to the values measured in
Section 8.2.3. If the data are not comparable, the analyst must review potential problem
areas and repeat the test.
8.3 The analyst must calculate method performance criteria and define the performance of the
laboratory for each spike concentration and parameter being measured.
8.3.1 Calculate upper and lower control limits for method performance as follows:
Upper control Limit (UCL) = R + 3s
Lower Control Limit (LCL) = R -3s
where R and s are calculated as in Section 8.2.3. The UCL and LCL can be used to
construct control charts 6 that are useful in observing trends in performance.
8.3.2 The laboratory mUst develop and maintain separate accuracy statements of laboratory
performance for wastewater samples. An accuracy statement for the method is defined
as R ± s. The accuracy statement should be developed by the analysis of four aliquots
of wastewater as described in Section 8.2.2, followed by the calculation of R and s.
Alternatively, the analyst may use four wastewater data points gathered through the
requirement for continuing quality control in Section 8.4. The accuracy statements
should be updated regularly. 6
8.4 The laboratory is required to collect in duplicate a portion of their samples to monitor spike
recoveries. The frequency of spiked sample analysis must be at least 10% of all samples or one
sample per month, whichever is greater. One aliquot of the sample must be spiked and analyzed
as described in Section 8.2. If the recovery for a particular compound does not fall within the
control limits for method performance, the results reported for that compound in all samples
processed as part of the same set must be qualified as described in Section 13.3. The laboratory
should monitor the frequency of data so qualified to ensure that it remains at or below 5%.
8.5 Before processing any samples, the analyst should demonstrate through the analysis of a 1-L
aliquot of reagent water that all glassware and reagent interferences are under control. Each time
a set of samples is extracted or there is a change in reagents, a laboratory reagent blank should
be processed as a safeguard against laboratory contamination.
8.6 it is recommended that the laboratory adopt additional quality assurance practices for use with this
method. The specific practices that are most productive depend upon the needs of the laboratory
and the nature of the samples. Field duplicates may be analyzed to monitor the precision of the
405
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Method 633.1
sampling technique. When doubt exists over the identification of a peak on the chromatogram,
confirmatory techniques such as gas chromatography with a dissimilar column, specific element
detector, or mass spectrometer must be used. Whenever possible, the laboratory should perform
analysis of standard reference materials and participate in relevant performance evaluation studies.
9. SAMPLE COLLEC77ON, PRESERVATiON, AND HANDLING
9.1 Grab samples must be collected in glass containers. Conventional sampling practices 7 should be
followed; however, the bottle must not be prerinsed with sample before collection. Composite
samples should be collected in refrigerated glass containers in accordance with the requirements
of the program. Automatic sampling equipment must be as free as possible of plastic and other
potential sources of contfimination.
9.2 The samples must be iced or refrigerated at 4°C from the time of collection until extraction.
9.3 Adjust the pH of the sample to 6 to S with 6N sodium hydroxide or 6N sulfuric acid immediately
after sampling.
10. SAMPLE EXTRACTION
10.1 Mark the water meniscus on the side of the sample bottle for later determination of sample
volume. Pour the entire sample into a 2-L separatory funnel. Check the pH of the sample with
wide range pH paper and adjust to 6 to 8 with 6N sodium hydroxide or 6N sulfuric acid.
10.2 Add 60 mL of methylene chloride to the sample bottle, seal, and shake 30 seconds to rinse the
inner wails. Transfer the solvent to the separatory funnel and extract the sample by shaking the
funnel for 2 minutes with periodic venting to release excess pressure. Allow the organic layer to
separate from the water phase for a minimum of 10 minutes. If the emulsion interface between
layers is more than one-third the volume of the solvent layer, the analyst must employ mechanical
techniques to complete the phase separation. The optimum technique depends upon the sample,
but may include stirring, filtration of the emulsion through glass wool, centrifugation, or other
physical methods. Collect the methylene chloride extract in a 250-mL Erlenmeyer flask.
10.3 Add a second 60-mL volume of methylene chloride to the sample bottle and repeat the extraction
procedure a second tune, combining the extracts in the Erlenmeyer flask. Perform a third
extraction in the same manner.
10.4 Assemble a Kuderna-Danish (K-D) concentrator by attaching a 10-mL concentrator tube to a
500-mL evaporative flask. Other concentration devices or techniques may be used in place of the
K-D if the requirements of Section 8.2 are met.
10.5 Pour the combined extract through a drying column containing about 10 cm of anhydrous sodium
sulfate, and collect the extract in the K-I) concentrator. Rinse the Erlenmeyer flask and column
with 20 to 30 mL of methylene chloride to complete the quantitative transfer. Once the flask rinse
has passed through the drying column, rinse the column with 30 to 40 mL of methylene chloride.
10.6 Add one or two clean boiling chips to the evaporative flask and attach a three-ball Snyder column.
Prewet the macro Snyder column by adding about 1 mL methylene chloride to the top. Place the
K-I) apparatus on a hot water bath, 60 to 65°C, so that the concentrator tube is partially immersed
in the hot water, and the entire lower rounded surface of the flask is bathed with hot vapor.
Adjust the vertical position of the apparatus and the water temperature as required to complete the
406
-------
Method 633.1
concentration in 15 to 20 minutes. At the proper rate of distillation, the balls of the column will
actively chatter but the chambers will not flood with condensed solvent. When the apparent
volume of liquid reaches 1 mL, remove the K-D apparatus and allow it to drain and cool for at
least 10 minutes.
10.7 Remove the macro-Snyder column and rinse the flask and its lower joint into the concentrator tube
with 1 to 2 mL of methylene chloride. Add one or two clean boiling chips and attach a two-ball
micro-Snyder column to the concentrator tube. Prewet the micro-Snyder column with methylene
chloride and concentrate the solvent extract as before. When an apparent volume of 0.5 mL is
reached, or the solution stops boiling, remove the K-D apparatus and allow it to drain and cool
for 10 minutes.
10.8 Remove the micro-Snyder column and adjust the volume of the extract to 1.0 mL with methylene
chloride. Stopper the concentrator tube and store refrigerated if further processing will not be
performed immediately. If the extract is to be stored longer than 2 days, transfer the extract to
a screw-capped vial with a VFFE-lined cap. If the sample extract requires no further cleanup,
proceed with solvent exchange to acetone as described in Section 10.9. If the sample requires
cleanup, proceed to Section 11.
10.9 Add one or two clean boiling chips to the concentrator tube along with 10 mL of acetone. Attach
the two-ball macro Snyder column and prewet the column with about 1 mL of acetone. Adjust
the temperature of the water bath -to 85 to 95°C. Concentrate the solvent extract as before to an
apparent volume of 0.5 mL and allow it to drain and cool for 10 minutes. Add a second 10 mL
of acetone to the concentrator tube and repeat the concentration procedure a second time. Adjust
the final volume of the extract to 1.0 mL with acetone.
10.10 Determinà the original sample volume by refilling the sample bottle to the mark and transferring
the water to a 1000-mL graduated cylinder. Record the sample volume to the nearest 5 mL.
11. CLEANUP AND SEPARA liON
11.1 Cleanup procedures may not be necessary for a relatively clean sample matrix. The cleanup
procedure recommended in this method has been used for the analysis of various clean waters and
industrial effluents. If particular circumstances demand the use of an alternative cleanup
procedure, the analyst must determine the elution profile and demonstrate that the recovery of
each compound of interest is no less than 85%.
11.2 The following Florisil cleanup procedure has been demonstrated to be applicable to the four
neutral nitrogen pesticides listed in Table 1.
11.2.1 Slurry 20 g of Florisil in 100 mL of ethyl ether and 400 &L of reagent water. Transfer
the slurry to a chromatographic column (Florisil may be retained with a plug of glass
wool). Allow the solvent to elute from the column until the Florisil is almost exposed
to the air. Wash the column with 25 mL of petroleum ether. Use a column flow rate
of 2 to 2.5 mL/rnin throughout the wash and elution profiles. Add an additional 50 mL
of petroleum ether to the head of the column.
11.2.2 Quantitatively transfer the sample from Section 10.8 to the petroleum ether suspended
over the column. Allow the solvent to elute from the column until the Florisil is almost
exposed to the air. Elute the column with 50 mL of 50% ethyl ether in petroleum ether.
Discard this fraction.
407
-------
Method 633.1
11.2.3 Elute the column with 50 mL of 100% ethyl ether (Fraction 1) and collect in a K-D
apparatus. Repeat procedure With 50 mL 6% acetone in ethyl ether (Fraction 2), 50 inL
15% acetone in ethyl ether (Fraction 3), 50 mL 50% acetone in ethyl ether (Fraction 4),
and 100 mL 100% acetone (Fraction 5), collecting each in a separate K-D apparatus.
The elution patterns for the neutral nitrogen compounds are shown in Table 1.
Concentrate each fraction to 1 mL as described in Section 10.6 and 10.7. The fractions
may be combined before concentration at the discretion of the analyst. Solvent exchange
Fraction 1 to acetone as described in Section 10.9 if the fractions are not combined.
11.2.4 Proceed with gas chromatographic analysis.
12. GAS CHROMATOGRAPHY
12.1 Table 2 summarizes the recommended operating conditions for the gas chromatograph. Included
in this table are estimated retention times and method detection limits that can be achieved by this
method. Examples of the separations achieved by Column 1 and Column 2 are shown in Figures
1 and 2. Other packed columns, chromatographic conditions, or detectors may be used if the
requirements of Section 8.2 are met. Capillary (open-tubular) columns may also be used if the
relative standard deviations of responses for replicate injections are demonstrated to be less than
6% and the requirements of Section 8.2 are met.
12.2 Calibrate the gas chromatographic system daily as described in Section 7.
12.3 If the internal standard approach is being used, the analyst must not add the internal standard to
the sample extracts until immediately before injection into the instrument. Mix thoroughly.
12.4 Inject 1 to 5 pL of the sample extract using the solvent flush technique. 8 Record the volume
injected to the nearest 0.05 L, and the resulting peak sizes in area or peak height units. An
automated system that consistently injects a constant volume of extract may also be used.
12.5 The width of the retention-time window used to make identifications should be based upon
measurements of actual retention-time variations of standards over the course of a day. Three
times the standard deviation of a retention time for a compound can be used to calculate a
suggested window size; however, the experience of the analyst should weigh heavily in the
interpretation of chromatograms.
12.6 If the response for the peak exceeds the working range of the system, dilute the extract and
reanalyze.
12.7 If the measurement of the peak response is prevented by the presence of interferences, further
cleanup is required.
13. CALCULA TIONS
13.1 Determine the concentration of individual compounds in the sample.
13.1.1 If the external standard calibration procedure is used, calculate the amount of material
injected from the peak response using the calibration curve or calibration factor in
Section 7.2.2. The concentration in the sample can be calculated as follows:
408
-------
Method 633.1
Equation 2
Concentration, igIL =
(V,)(V )
where
A = Amount of material injected, in ng
V 1 = Volume of extract injected, in pL
V 2 = Volume of total extract, in pL
V 2 = Volume of water extracted, in mL
13.1.2
If the internal standard calibration procedure was used, calculate the concentration in
the sample using the response factor (RF) determined in Section 7.3.2 as follows:
Equation 3
Concentration, tgIL = (A XRF)(v.)
where
A, = Response for parameter to be measured
A = Response for the internal standard
= Amount of internal standard added to each extract, in g
V 0 = Volume of water extracted, in L
13.2 Report results in micrograms per liter without correction for recovery data. When duplicate and
spiked samples are analyzed, report all data obtained with the sample results.
13.3 For samples processed as part of a set where the laboratory spiked sample recovery falls outside
of the control limits in Section 8.3, data for the affected compounds must be labeled as suspect.
14. GC/MS CONRRMA TFON
14.1 It is recommended that GC/MS techniques be judiciously employed to support qualitative
identifications made with this method. The mass spectrometer should be capable of scanning the
mass range from 35 amu to a mass 50 amu above the molecular weight of the compound. The
instrument must be capable of scanning the mass range at a rate to produce at least 5 scans per
peak but not to exceed 7 seconds per scan utilizing a 70 V (nominal) electron energy in the
electron impact ionization mode. A GC to MS interface constructed of all glass or glass-lined
materials is recommended. When using a fused-silica capillary column, the column outlet should
be threaded through the interface to within a few mm of the entrance to the source ionization
chamber. A computer system should be interfaced to the mass spectrometer that allows the
continuous acquisition and storage on machine-readable media of all mass spectra obtained
throughout the duration of the chromatographic program.
409
-------
Method 633.1
14.2 Gas chromatographic columns and conditions should be selected for optimum separation and
performance. The conditions selected must be compatible with standard GC/MS operating
practices. Chromatographic tailing factors of less than 5.0 must be achieved.’ 0
14.3 At the beginning of each day that confirmatory analyses are to be performed, the GC/MS system
must be checked to see that all DFTPP performance criteria are achieved. 9
14.4 To confirm an identification of a compound, the background-corrected mass spectrum of the
compound must be obtained from the sample extract and compared with a mass spectrum from
a stock or calibration standard analyzed under the same chromatographic conditions. It is
recommended that at least 50 ng of material be injected into the GCIMS. The criteria below must
be met for qualitative confirmation.
14.4.1 The molecular ion and all other ions that are present above 10% relative abundance in
the mass spectrum of the standard must be present in the mass spectrum of the sample
with agreement to ±10%. For example, if the relative abundance of an ion is 30% in
the mass spectrum of the standard, the allowable limits for the relative abundance of that
ioninthemassspectrumforthesamplewouldbe20to40%.
14.4.2 The retention time of the compound in the sample must be Within 6 seconds of the same
compound in the standard solution.
14.4 3 Compounds that have very similar mass spectra can be explicitly identified by GC/MS
only on the basis of retention time data.
14.5 Where available, chemical ionization mass spectra may be employed to aid in the qualitative
identification process.
14.6 Should these MS procedures fail to provide Satisfactory results, additional steps may be taken
before reanalysis. These may include the use of alternative packed or capillary GC columns or
additional cleanup (Section 11).
15. METHOD PERFORM4NCE
15.1 The method detection limit (MDL) is defined as the minimum concentration of a substance that
can be measured and reported with 99% confidence that the value is above zero.” The MDL
concentrations listed in Table 2 were obtained using reagent water.’ Similar results were achieved
using representative wastewaters.
15.2 This method has been tested for linearity of recovery from spiked reagent water and has been
demonstrated to be applicable over the concentration range from 10 x MDL to 1000 X MDL.
15.3 In a single laboratory, Battelle’s Columbus Laboratories, using spiked wastewater samples, the
average recoveries presented in Table 3 were obtained after Florisil cleanup. Seven replicates of
each of two different wastewaters were spiked and analyzed. The standard deviation of the
percent recovery is also included in Table 3.’
410
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Method 633.1
References
1. “Development of Methods for Pesticides in Wastewaters”, EPA Contract Report 68-03-2956 (In
preparation).
2. ASTM Annual Book of Standards, Part 31, D3694, “Standard Practice for Preparation of Sample
Containers and for Preservation”, American Society for Testing and Materials, Philadelphia,
Pennsylvania, p. 679, 1980.
3. “Carcinogens - Working with Carcinogens”, Department of Health, Education, and Welfare,
Public Health Service, Center for Disease Control, National Institute for Occupational Safety and
Health, Publication No. 77-206, August, 1977.
4. “OSHA Safety and Health Standards, General Industry”. (29 CFR 1910), Occupational Safety and
Health Administration, OSHA 2206 (Revised, January, 1976).
5. “Safety in Academic Chemistry Laboratories”, American Chemical Society Publication,
Committee on Chemical Safety, 3rd Edition, 1979.
6. “Handbook for Analytical Quality Control in Water and Wastewater Laboratories”,
EPA-600/4-79-019, U. S. Environmental Protection Agency, Environmental Monitoring and
Support Laboratory - Cincinnati, Ohio, March, 1979.
7. ASTM Annual Book of Standards, Part 31, D3370, “Standard Practice for Sampling Water,”
American Society for Testing and Materials, Philadelphia, Pennsylvania, p. 76, 1980.
8. Burke, J.A., “Gas Chromatography for Pesticide Residue Analysis; Some Practical Aspects,”
Journal of the Association of Official Analytical Chemists, 48, 1037 (1965).
9. Eichelberger, J.W., Harris, L.E., and Budde, W.L., “Reference Compound to Calibrate Ion
Abundance Measurement in Gas Chromatography-Mass Spectrometry”, Analytical Chemistry, 47,
995 (1975).
10. McNair, H.M., and Bonelli, E.J., Basic Chromatography, Consolidated Printing, Berkeley,
California, 52 (1969).
11. Glaser, l.A., et al., “Trace Analysis for Wastewaters”, Environmental Science and Technology,
15, 1426 (1981).
411
-------
Method 633.1
Table 1. Elution Characteristics of the Neutral Nitrogen Compounds on 2%
Deactivated Florisil
Elution in Specified Fraction(a)
Parameter Fl F2 F3 F4 F5
Fenarimol X X
MGK264 X X
MGK326 X X
Pronamide X
(a) Elution solvents are 50 mL each of the following:
Fl = 100% ethyl ether
F2 =6% acetone in ethyl ether
F3 = 15% acetone in ethyl ether
F4 = 50% acetone in ethyl ether
F5 = 100% acetone (100 mL)
Table 2. Chromatographic Conditions and Method Detection Limits
Retention Time (mu.) MDL
Parameter Column 1 Column 2 (pg/Li
Pronamide 19.9 22.0 4
MGK 326 21.9 23.8 6
MGK264 23.0 and 25.5 and 2
23.5(a) 27.5(a)
Fenarimol 30.6 32.2 4
Column 1 conditions: Supelcoport (100/120 mesh) coated with 3% SP-2250 packed in a 1.8 m long by
2 mm ID glass column with helium carrier gas at a flow rate of 30 mL/min. Column temperature is
programmed from 80°C to 300°C at 8°C/mm with a 4 minute hold at each extreme, injector temperature
is 250°C and detector is 300°C. Alkali flame detector at bead voltage of 16 V.
Column 2 conditions: Supelcoport (1001120 mesh) coated with 3% SP-2100 packed in a 1.8 m long by
2mm lDglasscolumnwithheliumcarriergasataflowrateof30mL/min. Allotherconditionsasfor
Column 1.
(a) Two isomers of MGK 264 are resolved from each other.
412
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Method 633.7
Table 3. Single-Laboratory Accuracy and Precision(a)
Spike Mean Standard Number
Sample Background, Level, Recovery, Deviation, of
Parameter Type(b) pg/L(c) pg/L Replicates
Fenariniol 1 1.8 20 98 4 7
2 ND 500 96 4 7
MGK264 1 ND 20 96 23 7
2 ND 500 74 4 7
MGK326 1 ND 20 108 7 7
2 ND 500 95 4 7
Pronanude 1 ND 20 102 5 7
2 210 500 86 3 7
(a) Column 1 conditions were used.
(b) 1 = Low level relevant industrial effluent
2 = High level relevant industrial effluent
(c) ND = Not detected
413
-------
Method - .1
Fenanmol
/
MGK 326
Pronamide
MGK 264
//
/ I I I I I I I I I I I I I I
0 17.0 19.0 21.0 23.0 25.0 27.0 29.0 31.0 33.0 35.0
Retention Time (minutes)
Figure 1. GC-AFD Chromatogram of 100 ng Each of the Neutral Nitrogen
Compounds (Column 1).
414
-------
Method aa I
Figure 2. GC-AFD Chromatogram of 200 ng Each of the Neutral Nitrogen
Compounds (Column 2).
/ Pronamide
MGK 326
\
Fenarimol
MGK 264
I-.
0 21.0 24.0 27.0 30.0
33.0
Retention TIme (minutes)
A52-O -33
415
-------
Method 634
The Determination of
Thiocarbate Pesticides in
Municipal and Industrial
Waste waters
-------
Method 634
The Determination of Thiocarbate Pesticides in Municipal and
Industrial Waste waters
SCOPE AND APPLICA TION
1.1 This method covers the determination of certain thiocarbamate pesticides. The following
parameters can be determined by this method:
Parameter CAS No.
Butylate 2008-41-5
Cycloate 1134-23-2
EPTC 759-94-4
Molinate 2212-67-1
Pebulate 1114-71-2
Vernolate 1929-77-7
1.2 This is a gas chromatographic (GC) method applicable to the determination of the compounds
listed above in municipal and industrial discharges as provided under 40 CFR 136.1. Any
modification of this method beyond those expressly permitted shall be considered a major
modification subject to application and approval of alternative test procedures under 40 CFR 136.4
and 136.5.
1.3 The method detection limit (MDL, defined in Section 15) for each parameter is listed in Table 1.
The MDL for a specific wastewater may differ from those listed, depending upon the nature of
interferences in the sample matrix.
1.4 The sample extraction and concentration steps in this method are similar to other 600-series
methods. Thus, a single sample may be extracted to measure the compounds included in the
scope of the methods. When cleanup is required, the concentration levels must be high enough
to permit selecting aliquots, as necessary, in order to apply appropriate cleanup procedures.
1.5 This method is restricted to use by or under the supervision of analysts experienced in the use of
gas chromatography and in the interpretation of gas chromatograms. Each analyst must
demonstrate the ability to generate acceptable results with this method using the procedure
described in Section 8.2.
1.6 When this method is used to analyze unfamiliar samples for any or all of the compounds above,
compound identifications should be supported by at least one additional qualitative technique.
This method describes analytical conditions for a second gas chromatographic column that can be
used to confirm measurements made with the primary column. Section 14 provides gas
chromatograph/mass spectrometer (GC!MS) criteria appropriate for the qualitative confirmation
of compound identifications.
419
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Method 634
2. SUMMARY OF METHOD
21 A measured volume of sample, approximately 1 L, is extracted with methylene chloride using a
continuous extractor. The methylene chloride extract is dried and concentrated to 5.0 mL. Gas
chromatographic conditions are described which permit the separation and measurement of the
compounds in the extract by alkali flame detector (AFD) gas chromatography. 1
2.2 This method provides an optional silica gel column cleanup procedure to aid in the elimination
of interferences which may be encountered.
3. INTERFERENCES
3.1 Method interferences may be caused by contaminants in solvents, reagents, glassware, and other
sample-processing apparatus that lead to discrete artifacts or elevated baselines in gas
chromatograms. All reagents and apparatus must be routinely demonstrated to be free from
interferences under the conditions of the analysis by running laboratory reagent blanks as
described in Section 8.5.
3.1.1
Glassware must be scrupulously cleaned. 2 Clean all glassware as soon as possible after
use by thoroughly rinsing with the last solvent used in it. Follow by washing with hot
water and detergent and thorough rinsing with tap and reagent water. Drain dry, and
heat in an oven or muffle furnace at 400°C for 15 to 30 minutes. Thermally stable
materials, such as PCBs, may not be eliminated by this treatment. Thorough rinsing with
acetone and pesticide-quality hexane may be substituted for the heating. After drying and
cooling, seal and store glassware in a clean environment to prevent any accumulation of
dust or other contaminants. Store inverted or capped with aluminum foil.
3.1.1 The use of high-purity reagents and solvents helps to minimize interference problems.
Purification of solvents by distillation in all-glass systems may be required.
3.2 Matrix interferences may be caused by contaminants that are coextracted from the sample. The
extent of matrix interferences will vary considerably from source to source, depending upon the
natnre and diversity of the industrial complex or municipality being sampled. The cleanup
procedure in Section 11 can be used to overcome many of these interferences, but unique samples
may require additional cleanup approaches to achieve the MDL listed in Table 1.
4. SAFETY
4.1 The toxicity or carcinogenicity of each reagent used in this method has not been precisely defined;
however, each chemical compound should be treated as a potential health hazard. From this view-
point, exposure to these chemicals must be reduced to the lowest possible level by whatever means
available. The laboratory is responsible for maintaining a current awareness file of OSHA
regulations regarding the safe handling of the chemicals specified in this method. A reference file
of material data handling sheets should also be made available to all personnel involved in the
chemical analysis. Additional references to laboratory safety are available and have been
identified” for the information of the analyst.
5. APPARA TUS AND MA TERIALS
5.1 Sampling equipment, for discrete or composite sampling.
420
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Method 634
5.1 .1 Grab-sample bottle: Amber borosiicate or flint glass, l-L or 1-quart volume, fitted with
screw-caps lined with PTFE. Aluminum foil may be substituted for PTFE if the sample
is not corrosive. If amber bottles are not available, protect samples from light. The
container and cap liner must be washed, rinsed with acetone or methylene chloride, and
dried before use to minimize contamination.
5.1.2 Automatic sampler (optional): Must incorporate glass sample containers for the
collection of a minimum of 250 mL. Sample containers must be kept refrigerated at 4°C
and protected from light during compositing. If the sampler uses a peristaltic pump, a
minimum length of compressible silicone rubber tubing should be thoroughly rinsed with
methanol, followed by repeated rinsings with reagent water to minimize the potential for
contamination of the sample. An integrating flow meter is required to collect flow-
proportional composites.
5.2 Glassware. (All Specifications are suggested. Catalog numbers are included for illustration only.)
5.2.1 Continuous extractor: 2000-mL, Hirschberg-Wolf, (Paxton Woods Glass Shop #1029 or
equivalent).
5.2.2 Drying column: Chromatographic column 400 mm long by 10 mm ID.
5.2.3 Chromatographic column: 400 mm long by 19 mm JD with 250-mL reservoir at the top
and PTFE stopcock (Kontes K-420290 or equivalent).
5.2.4 Concentrator tube, Kuderna-Danish: 25-mL, graduated (Kontes K-570050-1025 or
equivalent). Calibration must be checked at the volumes employed in the test. A
ground-glass stopper is used to prevent evaporation of extracts.
5.2.5 Evaporative flask, Kuderna-Danish: 500-mL (Kontes K-570001-0500 or equivalent).
Attach to concentrator tube with springs.
5.2.6 Snyder column, Kuderna-Danish: Three-ball macro (Kontes K-503000-0121 or
equivalent).
5.2.7 Snyder column, Kuderna-Danish: Two-ball micro (Kontes K-569001-0219 or
equivalent).
5.2.8 Vials: Amber glass, 10- to 15-mL capacity with PTFE-lined screw-cap.
5.2.9 Volumetric flask: 5-mL with glass stopper.
5.2.10 Volumetric flask: 10-mL with glass stopper.
5.2.11 Graduated cylinder: 1000-mL.
5.3 Boiling chips: Approximately 10/40 mesh carborundum. Heat to 400°C for 4 hours or extract
in a Soxhiet extractor with methylene chloride.
5.4 Water bath: Heated, capable of temperature control (±2°C). The bath should be used in a hood.
5.5 Balance: Analytical, capable of accurately weighing to the nearest 0.0001 g.
5.6 Gas chromatograph: Analytical system complete with gas chromatograph suitable for on-column
injection and all required accessories including syringes, analytical columns, gases, detector, and
strip-chart recorder. A data system is recommended for measuring peak areas.
5.6.1 Column 1: 180 cm long by 2 mm ID glass, packed with 3% SP-2250 on Supelcoport
(100/120 mesh) or equivalent. This column was used to develop the method performance
421
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Method 634
statements in Section 15. Guidelines for the use of alternative columns are provided in
Section 12.1.
5.6.2 Column 2: 180 cm long by 2 mm ID glass, packed with 3% SP—l000 on Supelcoport
(100/120 mesh) or equivalent.
5.6.3 Detector: Alkali flame detector (AFD), sometimes referred to as a nitrogen-phosphorus
detector (NPD) or a thermionic-specific detector (TSD). This detector has proven
effective in the analysis of wastewaters for the compounds listed in the scope and was
used to develop the method performance statements in Section 15. Alternative detectors,
including a macs spectrometer, may be used in accordance with the provisions described
inSection 12.1.
6. REAGENTS
6.1 Reagent water: Reagent water is defined as a water in which an interferent is not observed at the
method detection limit of each parameter of interest.
6.2 Methylene chloride, methanol, petroleum ether, ethyl ether, toluene: distilled-in-glass quality or
equivalent. Ethyl ether must be free of peroxides as indicated by EM Quant test strips (available
from Scientific Products Co., Catalog No. P1126-8 and other suppliers). Procedures
recommended for removal of peroxides are provided with thetest strips.
6.3 Sodium sulfate: ACS, granular, anhydrous; heated in a muffle furnace at 400°C overnight.
6.4 Silica gel: Davision Grade 923, 100/200 mesh; activated by heating for 24 hours at 150°C.
6.5 6N sodium hydroxide: Dissolve 24.0 grams NaOH in 100 niL distilled water.
6.6 6N Sulfuric acid. Slowly add 16.6 niL concentrated H 2 S0 4 to 50 mL distilled water and dilute
to 100 niL with distilled water.
6.7 Stock standard solutions (1.00 g g/pl): Stock standard solutions can be prepared from pure
standard materials or purchased as certified solutions.
6.7.1 Prepare stock standard solutions by accurately weighing about 0.0100 g of pure material.
Dissolve the material in distilled-in-glass quality methanol and dilute to volume in a
10-niL volumetric flask. Larger volumes can be used at the convenience of the analyst.
if compound purity is certified at 96% or greater, the weight can be used without
correction to calculate the concentration of the stock standard. Commercially prepared
stock standards can be used at any concentration if they are certified by the manufacturer
or by an independent source.
6.7.2 Transfer the stock standard solutions into PTFE-sealed screw-cap bottles. Store at 4°C
and protect from light. Frequently check stock standard solutions for signs of
degradation or evaporation, especially just prior to preparing calibration standards from
them.
6.7.3 Stock standard solutions must be replaced after 6 months, or sooner if comparison with
check standards indicates a problem.
422
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Method 634
7. CALIBRATiON
7.1 Establish gas chromatographic operating parameters equivalent to those indicated in Table 1. The
gas chromatographic system may be calibrated using either the external standard technique
(Section 7.2) or the internal standard technique (Section 7.3).
7.2 External standard calibration procedure.
7.2.1 For each compound of interest, prepare calibration standards at a minimum of three
concentration levels by adding volumes of one or more stock standards to a volumetric
flask and diluting to volume with toluene. One of the external standards should be at a
concentration near, but above, the method detection limit. The other concentrations
should correspond to the expected range of concentrations found in real samples or
should define the working range of the detector.
7.2.2 Using injections of 2 to 5 L of each calibration standard, tabulate peak height or area
responses against the mass injected. The results can be used to prepare a calibration
curve for each parameter. Alternatively, the ratio of the response to the mass injected,
defined as the calibration factor (CF), can be calculated for each compound at each
standard concentration. If the relative standard deviation of the calibration factor is less
than 10% over the working range, the average calibration factor can be used in place of
a calibration curve. -
7.2.3 The working calibration curve or calibration factor must be verified on each working
shift by the measurement of one or more calibration standards. If the response for any
compound varies from the predicted response by more than ±10%, the test must be
repeated using a fresh calibration standard. Alternatively, a new calibration curve or
calibration factor must be prepared for that parameter.
7.3 Internal standard calibration procedure: To use this approach, the analyst must select one or more
internal standards similar in analytical behavior to the compounds of interest. The analyst must
further demonstrate that the measurement of the internal standard is not affected by method or
matrix interferences. Due to these limitations, no internal standard applicable to all samples can
be suggested, although carbazole has been used successfully in some instances.
7.3.1 Prepare calibration standards at a minimum of three concentration levels for each
parameter of interest by adding volumes of one or more stock standards to a volumetric
flask. To each calibration standard, add a known constant amount of one or more
internal standards, and dilute to volume with toluene. One of the standards should be at
a concentration near, but above, the method detection limit. The other concentrations
should correspond to the expected range of concentrations found in real samples, or
should define the working range of the detector.
7.3.2 Using injections of 2 to 5 ,LL of each calibration standard, tabulate the peak height or
area responses against the concentration for each compound and internal standard. Calcu-
late response factors (RF) for each compound as follows:
423
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Method 634
Equation I
RF ( A,)(C )
(A )(C ,)
where
A, Response jbr the parameter to be measured
A Response Jbr the Internal staàdard
C, = Concentration of the internal standard, In pgIL
C, Concentration of the parameter to be measured, in igIL
If the RF value over the working range is constant, less than 10% relative standard
deviation, the RF can be assumed to be invariant and the average RF can be used for
calculations. Alternatively, the results can be used to plot a calibration curve of response
ratios, A,/A,, against RF.
7.3.3 The working calibration curve or RF must be verified on each working shift by the
measurement of one or more calibration standards. If the response for any càmpound
varies from the predicted response by more than ± 10%, the test must be repeated using
a fresh calibration standard. Alternatively, a new calibration curve must be prepared for
that compound.
7.4 Before using any cleanup procedure, the analyst must process a series of calibration standards
through the procedure to validate elution patterns and the absence of interferences from the
reagents.
8. QuALITY CONTROL
8.1 Each laboratory using this method is required to operate a formal quality control program. The
minimum requirements of this program consist of an initial demonstration of laboratory capability
and the analysis of spiked samples as a continuing check on performance. The laboratory is
required to mthntain performance records to define the quality of data that are generated.
8.1.1 Before performing any analyses, the analyst must demonstrate the ability to generate
acceptable accuracy and precision with this method. This ability is established as
described in Section 8.2.
8.1.2 In recognition of the rapid advances occurring in chromatography, the analyst is
permitted certain options to improve the separations or lower the cost of measurements.
Eath time such modifications to the method are made, the analyst is required to repeat
the procedure in Section 8.2
8.1.3 The laboratory must spike and analyze a minimum of 10% of all samples to monitor
continuing laboratory performance. This procedure is described in Section 8.4.
8.2 To establish the ability to generate acceptable accuracy and precision, the analyst must perform
the following operations.
8.2.1 Select a representative spike concentration for each compound to be measured. Using
stock standards, prepare a quality control check sample concentrate in methanol, 1000
times more concentrated than the selected concentrations.
424
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Method 634
8.2.2 Using a pipette, add 1.00 mL of the check sample concentrate to each of a minimum of
four 1000-mi. aliquots of reagent water. A representative wastewater may be used in
place of the reagent water, but one or more additional aliquots must be analyzed to
determine background levels, and the spike level must exceed twice the background level
for the test to be valid. Analyze the aliquots according to the method beginning in
Section 10.
8.2.3 Calculate the average percent recovery (R), and the standard deviation of the percent
recovery (s), for the results. Wastewater background corrections must be made before
R and s calculations are performed.
8.2.4 Using the appropriate data from Table 2, determine the recovery and single-operator
precision expected for the method, and compare these results to the values measured in
Section 8.2.3. If the data are not comparable, the analyst must review potential problem
areas and repeat the test.
8.3 The analyst must calculate method performance criteria and define the performance of the
laboratory for each spike concentration and parameter being measured.
8.3.1 Calculate upper and lower control limits for method performance as follows:
Upper Control Limit (UCL) = R + 3s
Lower Control Limit (LCL) = R - 3s
where R and s are calculated as in Section 8.2.3. The UCL and LCL can be used to
construct control charts 6 that are useful in observing trends in performance.
8.3.2 The laboratory must develop and maintain separate accuracy statements of laboratory
performance for wastewater samples. An accuracy statement for the method is defined
as R ± s. The accuracy statement should be developed by the analysis of four aliquots
of wastewater as described in Section 8.2.2, followed by the calculation R and s. Mter-
nately, the analyst may use four wastewater data points gathered through the requirement
for continuing quality control in Section 8.4. The accuracy statements should be updated
regularly. 6
8.4 The laboratory is required to collect in duplicate a portion of their samples to monitor spike
recoveries. The frequency of spiked sample analysis must be at least 10% of all samples or one
sample per month, whichever is greater. One aliquot of the sample must be spiked and analyzed
as described in Section 8.2. If the recovery for a particular compound does not fall within the
control limits for method performance, the results reported for that compound in all samples
processed as part of the same set must be qualified as described in Section 13.3. The laboratory
should monitor the frequency of data so qualified to ensure that it remains at or below 5%.
8.5 Before processing any samples, the analyst should demonstrate through the analysis of a 1-L
aliquot of reagent water that all glassware and reagent interferences are under control. Each time
a set of samples is extracted or there is a change in reagents, a laboratory reagent blank should
be processed as a safeguard against laboratory contamination.
8.6 It is recommended that the laboratory adopt additional quality assurance practices for use with this
method. The specific practices that are most productive depend upon the needs of the laboratory
and the nature of the samples. Field duplicates may be analyzed to monitor the precision of the
425
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Method 634
sampling technique. When doubt exists over the identification of a peak on the chromatogram,
confirmatory techniques such as gas chromatography with a dissimilar column, specific element
detector, or mass spectrometer must be used. Whenever possible, the laboratory should perform
analysis of standard reference materials and participate in relevant performance evaluation studies.
9. SAMPLES COLLECTiON, PRESERVATION AND HANDUNG
9.1 Grab samples must be collected in glass containers. Conventional sampling practices 7 should be
followed; however, the bottle must not be prerinsed with sample before collection. Composite
samples should be collected in refrigerated glass containers in accordance with the requirements
of the program. Automatic sampling equipment must be as free as possible of plastic and other
potential sources of contamination.
9.2 The samples must be iced or refrigerated at 4°C from the time of collection until extraction.
9.3 Adjust the pH of the sample to 6 to 8 with 6N sodium hydroxide or 6N sulfuric acid immediately
after sampling.
10. SAMPLE EXTRACTiON
10.1 Assemble continuous extraction apparatus by placing 5 to 10 carborundum chips into the 500-mL
round-bottom flask and attaching to the extraction flask.
10.2 Add 400 mL methylene chloride to the extraction flask. Some methylene chloride should displace
into the round-bottom flask.
10.3 Mark the water meniscus on the side of the sample bottle for later determination of sample
volume. Pour the entire sample into the extraction flask and add sufficient distilled water to fill
extraction flask (2 L total volume aqueous phase).
10.4 CheckthepflofthesamplewithwiderangepHpaperandadjustto6to8with6Nsodium
hydroxide or 6N sulfuric acid.
10.5 Connect the stirring apparatus to the extraction flask without the fit touching the sample. Heat
the methylene chloride in the round-bottom flask to continuous reflux and continue heating for 30
minutes to 1 hour until fit is thoroughly wetted with methylene chloride.
10.6 Lower fit until it just touches the sample and start the stirring apparatus rotating.
10.7 Continuously extract sample for 18 to 24 hours.
10.8 Assemble a Kuderna-Danish (K-D) concentrator by attaching a 25-mL concentrator tube to a
500-mL evaporative flask. Other concentration devices or techniques may be used in place of the
K-D if the requirements of Section 8.2 are met.
10.9 Pour the extract from the round-bottom flask through a drying column containing about 10 cm of
anhydrous sodium sulfate, and collect the extract in the K-D concentrator. Rinse the flask and
column with 20 to 30 mL of methylene chloride to complete the quantitative transfer. Once the
flask rinse has passed through the drying column, rinse the column with 30 to 40 mL of
methylene chloride.
10.10 Add one or two clean boiling chips to the evaporative flask and attach a three-ball Snyder
column. Prewet the Snyder column by adding about 1 niL methylene chloride to the top.
Place the K-D apparatus on a hot water bath, 60 to 65°C, so that the concentrator tube
426
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Method 634
is partially immersed in the hot water, and the entire lower rounded surface of the flask
is bathed with hot vapor. Adjust the vertical position of the apparatus and the water
temperature as required to complete the concentration in 15 to 20 minutes. At the proper
rate of distillation, the balls of the column will actively chatter but the chambers will not
flood with condensed solvent. When the apparent volume of liquid reaches 1 mL,
remove the K-D apparatus and allow it to drain and cool for at least 10 minutes.
10.11 Remove the Snyder column and flask and adjust the volume of the extract to 5.0 mL with
methylene chloride. Stopper the concentrator tube and store refrigerated if further
processing will not be performed immediately. If the extract is to be stored longer than
2 days, transfer the extract to a screw-capped vial with a PTFE-lined cap. If the sample
extract requires no further cleanup, proceed with solvent exchange to toluene as described
in Section 10.12, and then to gas chromatographic analysis as described in Section 12.
If the sample requires cleanup, proceed to Section 11.
10.12 Add 2.5 mL of toluene and one or two clean boiling chips to the extract in the 25-mL
concentrator tube and attach a two ball micro-Snyder column. Place the K-D apparatus
in a hot water bath, 70 to 75°C. when the apparent volume of liquid reaches 2 to 2.5
mL. Remove the K-D apparatus and allow it to drain and cool for at least 10 minutes.
Transfer the sample to a 5 mL volumetric flask and dilute to 5-mL with toluene. Proceed
with gas chromatographic analysis.
10.13 Determine the original sample volume by refilling the sample bottle to the mark and
transferring the water to a 100-mL graduated cylinder. Record the sample volume to the
nearest 5 mL.
11. CLEANUP AND SEPARA T1ON
11.1 Cleanup procedures may not be necessary for a relatively clean sample matrix. The cleanup
procedure recommended in this method has been used for the analysis of various clean waters and
industrial effluents. If particular circumstances demand the use of an alternative cleanup
procedure, the analyst must determine the elution profile and demonstrate that the recovery of
each compound of interest is no less than 85%.
11.2 Add20gofsiicagelto amixtureof lOOmLofacetoneand 1.2mLofreagentwaterandstir
for 30 minutes on a stirring plate. Transfer the slurry to a chromatographic column (silica gel
may be retained with a plug of glass wool). Wash the column with 20 mL of methylene chloride
followed by 30 mL petroleum ether. Allow the solvent to elute from the column until the silica
gel is almost exposed to the air. Discard washings. Use a column flow rate of 2 to 2.5 mL/min
throughout the wash and elution profiles. Add an additional 50 mL of petroleum ether to the head
of the column.
11.3 Add the extract from Section 10.12 to the petroleum ether suspended above column. Allow the
solvent to elute from the column until the silica gel is almost exposed to the air. Elute the column
with 25 mL of petroleum ether (Fl). Discard this fraction.
11.4 Elute the column with 100 mL of 50% ethyl ether in petroleum ether and collect in a K-D
apparatus. Alternatively, separate fractions may be collected or combined at the discretion of the
analyst. The elution profile of these compounds from silica gel is given in Table 3.
427
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Method 634
11.5 Concentrate the fraction to less than 5-mL after addition of 2.5 mL toluene as described in Section
10.12. Transfer sample to a 5-mL volumetric flask and dilute to 5 mL with toluene. Proceed with
gas chromatographic analysis.
12. GAS CHROMATOGRAPHY
12.1 Table 1 summarizes the recommended operating conditions for the gas chromatograph. Included
in this table are estimated retention times and method detection limits that can be achieved by this
method. An example of the separations achieved by Column 1 and Column 2 are shown in
Figures 1 and 2. Other packed columns, chromatographic conditions, or detectors may be used
if the requirements of Section 8.2 are met. Capillary (open-tubular) columns may also be used
if the relative standard deviations of responses for replicate injections are demonstrated to be less
than 6% and the requirements of Section 8.2 are met.
12.2 Calibrate the gas chromatographic system daily as described in Section 7.
12.3 If an internal standard approach is being used, the analyst must not add the internal standard to
sample extracts until immediately before injection into the instrument. Mix thoroughly
12.4 Inject 2 to 5 L of the sample extract using the solvent-flush technique.’ Record the volume
injected to the nearest 0.05 giL, and the resulting peak sizes in area or peak height units.
12.5 The width of the retention time window used to make identifications should be based upon
measurements of actual retention-time variations of standards over the course of a day. Three
times the standard deviation of a retention-time for a compound can be used to calculate a
suggested window size; however, the experience of the analyst should weigh heavily in the
interpretation of chromatograms.
12.6 if the response for the peak exceeds the working range of the system, dilute the extract and
reanalyze.
12.7 If the measurement of the peak response is prevented by the presence of interferences, further
cleanup is required.
13. CALCULATiONS
13.1 Determine the concentration of individual compounds in the sample.
13.1.1 If the external standard calibration procedure is used, calculate the amount of material
injected from the peak response using the calibration curve or calibration factor in Section
7.2.2. The concentration in the sample can be calculated as follows:
428
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Method 634
Equation 2
Concentration, pgIL ( A)(V 1 )
(V )(V,)
where
A = Amount of material injected, in ng
V 1 = Volume of extract injected, in ,zL
V 1 = Volume of total extract, in 1 iL
V 5 = Volume of water extracted, in niL
13.1.2 If the internal standard calibration procedure was used, calculate the concentration in the
sample using the response factor (RF) determined in Section 7.3.2 as follows:
Equation 3
Concentration, g/L =
(A ,)(RF)(V 0 )
.
where
A =
4, =
I ,
V 0 =
Response for parameter to be measured
Response for the internal standard
Amount of internal standard added to each extract,
Volume of water extracted, in L
in
g
13.2 Report results in micrograms per liter without correction for recovery data. When duplicate and
spiked samples are analyzed, report all data obtained with the sample results.
13.3 For samples processed as part of a set where the laboratory spiked sample recovery falls outside
of the control limits in Section 8.3, data for the affected compounds must be labeled as suspect.
14. GC/MS CONFIRMATION
14.1 It is recommended that GC/MS techniques be judiciously employed to support qualitative
identifications made with this method. The mass spectrometer should be capable of scanning the
mass range from 35 amu to a mass 50 amu above the molecular weight of the compound. The
instrument must be capable of scanning the mass range at a rate to produce at least 5 scans per
peak but not to exceed 7 seconds per scan utilizing a 70 V (nominal) electron energy in the
electron impact ionization mode. A (iC to MS interface constructed of all glass or glass-lined
materials is recommended. When using a fused-silica capillary column, the column outlet should
be threaded through the interface to within a few millimeters of the entrance to the source
ionization chamber. A computer system should be interfaced to the mass spectrometer that allows
the continuous acquisition and storage on machine-readable media of all mass spectra obtained
throughout the duration of the chromatographic program.
14.2 Gas chromatographic columns and conditions should be selected for optimum separation and
performance. The conditions selected must be compatible with standard GC(MS operating
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Method 634
practices. Chromatographic tailing factors of less than 5.0 must be achieved. The calculation of
tailing factors is illustrated in Method 625.
14.3 At the beginning of each day that confirmatory analyses are to be performed, the GCIMS system
must be checked to see that all DFTPP performance criteria are achieved. 9
14.4 To confirm an identification of a compound, the background-corrected mass spectrum of the
compound must be obtained from the sample extract and compared with a mass spectrum from
a stock or calibration standard analyzed under the same chromatographic conditions. It is
recommended that at least 50 ng of material be injected into the GC/MS. The criteria below must
be met for qualitative confirmation.
14.4.1 The molecular ion and all other ions that are present above 10% relative abundance in
the mass spectrum of the standard must be present in the mass spectrum of the sample
with agreement to ± 10%. For example, if the relative abundance of an ion is 30% in
the mass spectrum of the standard, the allowable limits for the relative abundance of that
loninthemass spectrum for thesamplewould be20to 40%.
14.4.2 The retention time of the compound in the sample must be within 30 seconds of the same
compound in the standard solution.
14.4.3 Compounds that have very similar mass spectra can be explicitly identified by GCIMS
only on the basis of retention-time data.
14.5 Where available, chemical ionimtion mass spectra may be employed to aid in the qualitative
identification process.
14.6 Should these MS procedures fail to provide satisfactory results, additional steps may be taken
before reanalysis. These may include the use of alternative packed or capillary GC columns or
additional cleanup (Section 11).
15. METHOD PERFORMANCE
15.1 The method detection limit (MDL) is defined as the minimum concentration of a substance that
can be measured and reported with 99% confidence that the value is above zero. The MDL
concentrations listed in Table 1 were obtained using reagent water.’
15.2 This method has been tested for linearity of recovery from spiked reagent water and has been
demonstrated to be applicable over the concentration range from 10 X MDL to 1000 X MDL.
15.3 In a single laboratory, Battelle’s Columbus Laboratories, using spiked wastewater samples, the
average recoveries presented in Table 2 were obtained. Seven replicates of each of two different
wastewaters were spiked and analyzed. The standard deviation of the percent recovery is also
included in Table 2.
4 O
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Method 634
References
1. “Development of Methods for Pesticides in Wastewaters ”. EPA Contract Report 68-03-2956 (In
preparation).
2. ASTM Annual Book of Standards, Part 31, D3694, “Standard Practice for Preparation of Sample
Containers and for Preservation”, American Society for Testing and Materials, Philadelphia,
Pennsylvania, p. 679, 1980.
3. “Carcinogens - Working with Carcinogens”, Department of Health, Education, and Welfare,
Public Health Service, Center for Disease Control, National Institute for Occupational Safety and
Health, Publication No. 77-206, August, 1977.
4. “OSHA Safety and Health Standards, General Industry” (29 CFR 1910), Occupational Safety and
Health Administration, OSHA 2206 (Revised, January, 1976).
5. Safety in Academic Chemistry Laboratories. American Chemical Society Publication, Committee
on Chemical Safety, 3rd Edition, 1979.
6. “Handbook for Analytical Quality Control in Water and Wastewater Laboratories”,
EPA-600/4-79-019, U. S. Environmental Protection Agency, Environmental Monitoring and
Support Laboratory - Cincinnati, Ohio 45268, March, 1979.
7. ASTM Annual Book of Standards, Part 31, D3370, “Standard Practice for Sampling Water”,
American Society for Testing and Materials, Philadelphia, Pennsylvania, p. 76, 1980.
8. Burke, J.A., “Gas Chromatography for Pesticide Residue Analysis; Some Practical Aspects,”
Journal of the Association of Official Analytical Chemists, 48, 1037 (1965).
9. Eichelberger. J.W., Harris, L.E., and Budde, W.L., “Reference Compound to Calibrate Ion
Abundance Measurement in Gas Chromatography - Mass Spectrometry”, Analytical Chemistry,
47, 995 (1975).
431
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Method 634
Table 1. Chromatographic Conditions and Method Detection Limits
Retention Time (minutes) Method
Detection Limit
Parameter Column 1 Column 2 (pg/L)
EPTC 12.8 17.9 0.9
Butylate 13.5 18.2 0.6
Vernolate 14.2 19.6 1.1
Pebulate 14.5 20.2 0.8
Molinate 16.6 23.8 0.6
Cycloate 17.5 24.2 1.6
Column 1 conditions: Supelcoport (100/120 mesh) coated with 3% SP-2250 packed in a 1.8 m long by
2 mm ID glass column with helium carrier gas at a flow rate of 30 mLImin. Column temperature is held
at 80°C for 4 minutes, programmed from 80°C to 300°C at 8°C/mm and held at 300°C for 4 minutes.
Column 2 conditions: Supelcoport (100/120 mesh) coated with 3% SP-2100 packed in a 1.8 m long by
2 mm ID glass column with helium carrier gas at a flow rate of 30 mL/min. Column temperature is held
at 80°C for 10 minutes, programmed from 80°C to 250°C at 8°C/min and held at 250°C for 10 minutes.
432
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Method 634
Tab’e 2. Single Laboratory Accuracy and Precision(a)
Relative
Average Standard Spike Number
Percent Deviation Level of Matrix
Parameter Recovery (pg/LI Analyses Type(b)
Butylate 80 18 5.0 7 1
95 7.2 50 7 1
Cycloate 93 16 5.0 7 1
95 7.3 50 7 1
EPTC 100 18 5.0 7 1
100 4.8 50 7 1
Molinate 87 17 5.0 7 1
93 8.4 50 7 1
Pehulate 97 26 5.0 7 1
98 5.7 50 7 1
Vernolate 93 18 5.0 7 1
96 10 50 7 1
(a) Column 1 conditions were used.
(b) 1 = secondary POTW effluent
433
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Method 634
Table 3. Elution Characteristics of the Thiocarbamates from 6% Deactivated
Silica Gel
Appearance in Specified Fraction(a)
Parameter Fl F2 F3 F4
Butylate x x
Cycloate x
EPTC x
Molinate x
Pebulate x
Vernolate x
Eluant composition by fraction:
(a) Fl =25 mL petroleum ether
F2 = 50 mL 6% ethyl ether in petroleum ether
F3 =50 mL 15% ethyl ether in petroleum ether
F4 =50 mL 50% ethyl ether in petroleum ether
434
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Method 634
___7, I
0 7.0
I I
9.0
Vemolate
EPTC
Butylate
Pebulate
/
Molinate
/
I I
I
I
I
I I
I I I I I I
11.0
13.0
15.0
17.0
19.0
21.0
Rate
ntlon TI
me
(minutes)
A .OC2 .23
Figure 1. GC-AFD Chromatogram of 200 ng of Each Thiocarbamate (Column 1).
Cycloate
/
435
-------
M.thod 634
Cycloate
Pebulate
Vemof ate
Mobnate
EPIC
1 I I I I I I I I_I 1 I I I 1—’ i I
0 16.0 17.0 18.0 19.0 20.0 21.0 22.0 23.0 24.0 25.0
Retention TIme (minutes)
A -O I -24
Figure 2. GC-AFD Chromatogram of 200 ng of Each Thiocarbamate (Column 2).
436
-------
Method 635
The Determination of Rotenone
in Municipal and Industrial
Waste waters
-------
Method 635
The Determination of Rotenone in Municipal and Industrial
Waste waters
1. SCOPE AND APPLICA TJON
1.1 This method covers the determination of rotenone pesticide. The following parameter can be
determined by this method:
Pa, metei CAS No.
Rotenone 83-79-4
1.2 This is a high performance liquid chromatographic (HPLC) method applicable to the determination
of the compound listed above in municipal and industrial discharges as provided under 40 CFR
136.1. Any modification of this method beyond those expressly permitted shall be considered a
major modification subject to application and approval of alternative test procedures under 40 CFR
136.4 and 136.5.
1.3 The method detection limit (MDL, defined in Section 14) for rotenone compound is listed in Table
1. The MDL for a specific wastewater may differ from those listed, depending upon the nature
of interferences in the sample matrix.
1.4 This method is restricted to use by or under the supervision of analysts experienced in the use of
liquid chromatography and in the interpretation of liquid chromatograms. Each analyst must
demonstrate the ability to generate acceptable results with this method using the procedure
described in Section 8.2.
1.5 When this method is used to analyze unfamiliar samples for the compound above, compound
identifications should be supported by at least one additional qualitative technique. This method
describes analytical conditions for a second liquid chromatographic column that can be used to
confirm measurements made with the primary column.
2. SUMMARY OF METHOD
2.1 A measured volume of sample, approximately 1 L, is extracted with methylene chloride using a
separatory funnel. Liquid chromatographic conditions are described which permit the separation
and measurement of the compounds in the extract by HPLC- UV.’
3. INTERFERENCES
3.1 Method interferences may be caused by contaminants in solvents, reagents, glassware, and other
sample-processing hardware that lead to discrete artifacts or elevated baselines in liquid
chromatograms. All of these materials must be routinely demonstrated to be free from
interferences under the conditions of the analysis by running laboratory reagent blanks as
described in Section 8.5.
3.1.1 Glassware must be scrupulously cleaned. 2 Clean all glassware as soon as possible after
use by thoroughly rinsing with the last solvent used in it. Follow by washing with hot
439
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Method 635
water and detergent and thoroughly rinsing with tap and reagent water. Drain dry, and
heat in an oven or muffle furnace at 400°C for 15 to 30 minutes. Some thermally stable
materials, such as PCBs, may not be eliminated by this treatment. Thorough rinsing with
acetone and pesticide-quality hexane may be substituted for the heating. After drying and
cooling, seal and store glassware in a clean environment to prevent accumulation of dust
or other contaminants. Store inverted or capped with aluminum foil.
3.1.2 The use of high-purity reagents and solvents helps to minimize interference problems.
Purification of solvents by distillation in all-glass systems may be required.
3.2 Matrix interferences may be caused by contaminants that are coextracted from the sample. The
extent of matrix interferences will vary considerably from source to source, depending upon the
nature and diversity of the industrial complex or municipality being sampled. The cleanup
procedure in Section 11 can be used to overcome these interferences, but unique samples may
requirb additional cleanup approaches to achieve the MDL listed in Table 1.
4.. SAFETY
4.1 The toxicity or carcinogemcity of each reagent used in this method has not been precisely defined;
however, each chemical compound should be treated as a potential health hazard. From this
viewpoint, exposure to these chemicals must be reduced to the lowest possible level by whatever
means available. The laboratory is responsible for maintaining a current awareness file of OSHA
regulations regarding the safe handling of the chemicals specified in this method. A reference file
of material data handling sheets should also be made available to all personnel involved in the
chemical analysis. Additional references to laboratory safety are available and have been
identifled 5 for the information of the analyst.
5. APPARA TUS AND MA TERIALS
5.1 Sampling equipment, for discrete or composite sampling.
5.1.1 Grab-sample bottle: Amber borosiicate or flint glass, 1-L or 1-quart volume, fitted with
screw-caps lined with PTFE. Aluminum foil may be substituted for PTFE if the sample
is not corrosive. If amber bottles are not available, protect samples from light. The
container and cap liner must be washed, rinsed with acetone or methylene chloride, and
dried before use to minimize contamination.
5.1.2 Automatic sampler (optional): Must incorporate glass sample containers for the
collection of a minimum of 250 mL. Sample containers must be kept refrigerated at 4°C
and protected from light during compositing. If the sampler uses a peristaltic pump, a
minimum length of compressible silicone rubber tubing may be used. Before use,
however, the compressible tubing should be thoroughly rinsed with methanol, followed
by repeated rinsings with distilled water to minimize the potential for contamination of
the sample. An integrating flow meter is required to collect flow-proportional composites.
5.2 Glassware. (All specifications are suggested. Catalog numbers are included for illustration only.)
5.2.1 Separatory funnel: 2000-mL, with PTFE stopcock.
5.2.2 Drying column: Chromatographic column 400 mm long by 10 mm ID with coarse fit.
440
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Method 635
5.2.3 Chromatographic column: 400 mm long by 19 mm ID with 250-mL reservoir at the top
and PTFE stopcock (Kontes K-420290) or equivalent).
5.2.4 Concentrator tube, Kuderna-Danish: 25-mL, graduated (Kontes K-570050-2525 or
equivalent). Calibration must be checked at the volumes employed in the test. A
ground-glass stopper is used to prevent evaporation of extracts.
5.2.5 Evaporative flask, Kuderna-Danish: 250-mL (Kontes K-570001-0250 or equivalent).
Attach to concentrator tube with springs.
5.2.6 Snyder column, Kuderna-Danish: Three-ball macro (Kontes K-503000-0121 or
equivalent).
5.2.7 Snyder column, Kuderna-Danish: Two-ball micro (Kontes K-569001-0219 or
equivalent).
5.2.8 Vials: Amber glass, 5- to l0-mL capacity with PTFE-lined screw-cap.
5.2.9 Volumetric flask: 10-mL.
5.2.10 Erlenmeyer flask: 250-mL.
5.2.11 Graduated cylinder: 1000-mL.
5.3 Boiling chips: Approximately 10/40 mesh carborundum. Heat to 400°C for 4 hours or extract
in a Soxhlet extractor with methylene chloride.
5.4 Water bath: Heated, capable of temperature control (±2°C). The bath should be used in a hood.
5.5 Balance: Analytical, capable of accurately weighing to the nearest 0.0001 g.
5.6 Liquid chromatograph: Analytical system complete with liquid chromatograph and all required
accessories including syringes, analytical columns, detector, and strip-chart recorder. A data
system is recommended for measuring peak areas.
5.6.1 Pump: Isocratic pumping system, constant flow.
5.6.2 Column 1: Normal-phase column, 5 Zorbax-CN, 250 mm long by 4.6 mm II) or
equivalent.
5.6.3 Column 2: Reversed-phase column, 5 j Spherisorb-ODS, 250 mm tong by 4.6 mm ID
or equivalent.
5.6.4 Detector: Ultraviolet absorbance detector, 254 nm.
6. REAGENTS
6.1 Reagent water: Reagent water is defined as a water in which an interferent is not observed at the
method detection limit of each parameter of interest.
6.2 Methylene chloride, methanol, acetothtrile, acetone, hexane: Distilled-in-glass quality or
equivalent.
6.3 Sodium sulfate: ACS, granular, anhydrous; heated in a muffle furnace at 400°C overnight.
6.4 iN sulfuric acid.
6.5 iN sodium hydroxide.
6.6 Silica gel, Davison grade 923, 100-200 mesh, dried for 12 hours at 150°C.
441
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Metho4 635
6.7 Stock standard solutions (1.00 pg/L): Stock standard solutions can be prepared from pure
standard materials or purchased as certified solutions.
6.7.1 Prepare stock standard solutions by accurately weighing about 0.0100 g of pure material.
Dissolve the material in distilled-in-glass quality methylene chloride for analyses
performed using Column land methanol for analyses performed using Column 2. Dilute
to volume in a 10-niL volumetric flask. Larger volumes can be used at the convenience
of the analyst. lfcompoundpurityis certified at 96% or greater, the weight can be used
without correction to calculate the concentration of the stock standard. Commercially
prepared stock standards can be used at any concentration if they are certified by the
manufacturer or by an independent source.
6.7.2 Transfer the stOck standard solutions into PTFE-sealed screw-cap bottles. Store at 4°C
and protect from light. Stock standard solutions should be checked frequently for signs
of degradation or evaporation, especially just prior to preparing calibration standards
from them.
6.7.3 Stock standard solutions must be replaced after 6 months, or sooner if comparison with
check standards indicates a problem.
7. CALIBRATION
7.1 Establish liquid chromatographic operating parameters equivalent to those indicated in Table 1.
The liquid diromatographic system can be calibrated using the external standard technique (Section
7.2) or the internal standard technique (Section 7.3).
7.2 External standard calibration procedure:
7.2.1 For each compound of interest, prepare calibration standards at a minimum of three
concentration levels by adding volumes of one or more stock standards to a volumetric
flask and diluting to volume with 50/50 hexane/methylene chloride for Column 1
standards and acetonitrile for Column 2 standards. One of the external standards should
be at a concentration near, but above, the method detection limit. The other
concentrations should correspond to the expected range of concentrations found in real
samples or should define the working range of the detector.
7.2.2 Using injections of 5 to 20 pL of each calibration standard, tabulate peak height or area
responses against the mass injected. The results can be used to prepare a calibration
curve for each compound. Alternatively, the ratio of the response to the mass injected,
defined as the calibration factor (CF), can be calculated for each compound at each
standard concentration. If the relative standard deviation of the calibration factor is less
than 10% over the working range, linearity through the origin can be assumed and the
average calibration factor can be used in place of a calibration curve.
7.2.3 The working calibration curve or calibration factor must be verified on each working
shift by the measurement of one or more calibration standards. If the response for any
compound varies from the predicted response by more than ± 10%, the test must be
repeated using a fresh calibration standard. Alternatively, a new calibration curve or
calibration factor must be prepared for that compound.
442
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Method 635
7.3 Internal standard calibration procedure: To use this approach, the analyst must select one or more
internal standards similar in analytical behavior to the compounds of interest. The analyst must
further demonstrate that the measurement of the internal standard is not affected by method or
matrix interferences. Due to these limitations, no internal standard applicable to all samples can
be suggested.
7.3.1 Prepare calibration standards at a minimum of three concentration levels for each
compound of interest by adding volumes of one or more stock standards to a volumetric
flask. To each calibration standard, add a known constant amount of one or more
internal standards, and dilute to volume with 50/50 hexane/methylene chloride for
Column 1 standards and acetonitrile for Column 2 standards. One of the standards
should be at a concentration near, but above, the method detection limit. The other
concentrations should correspond to the expected range of concentrations found in real
samples, or should define the working range of the detector.
7.3.2 Using injections of 5 to 20 iL of each calibration standard, tabulate the peak height or
area responses against the concentration for each compound and internal standard.
Calculate response factors (RF) for each compound as follows:
Equation 1
= (A,)(C ,,)
(A (C,)
.
where
A, =
A,, =
C,, =
C, =
Response for
Response for
Concentration
Concentration
the parameter to be measured
the internal standard
of the internal standard, in g/L
of the parameter to be measured, in pg/L
If the RF value over the working range is constant, less than 10% relative standard
deviation, the RF can be assumed to be invariant and the average RF can be used for
calculations. Alternatively, the results can be used to pilot a calibration curve of
response ratios, A)A 1 against RF.
7.3.3 The working calibration curve or RF must be verified on each working shift by the
measurement of one or more calibration standards. If the response for any compound
varies from the predicted response by more than ±10%, the test must be repeated using
a fresh calibration standard. Alternatively, a new calibration curve must be prepared for
that compound.
7.4 Before using any cleanup procedure, the analyst must process a series of calibration standards
through the procedure to validate elution patterns and the absence of interferences from the
reagents.
443
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Method 635
8. QuALm’ CONTROL
8.1 Each laboratory using this method is required to operate a formal quality control program. The
minimum requirements of this program consist of an initial demonstration of laboratory capability
and the analysis of spiked samples as a continuing check on performance. The laboratory is
required to maintain performance records to define the quality of data that is generated.
8.1 .1 Before performing any analyses, the analyst must demonstrate the ability to generate
acceptable accuracy and precision with this method. This ability is established as
described in Section 8.2.
8.1.2 In recognition of the rapid advances occurring in chromatography, the analyst is
permitted certain options to improve the separations or lower the cost of measurements.
each time such modifications to the method are made, the analyst is required to repeat
the procedure in Section 8.2.
8.1.3 The laboratory must spike and analyze a minimum of 10% of all samples to monitor’
continuing laboratory performance. This procedure is described in Section 8.4.
8.2 To establish the ability to generate acceptable accuracy and precision, the analyst must perform
the following operations.
8.2.1 Select a representative spike concentration for each compound to be measured. Using
stock standards, prepare a quality control check sample concentrate in methylene
chloride, 1000 times more concentrated than the selected concentrations.
8.2.2 Using a pipette, add 1.00 mL of the check sample concentrate to each of a minimum of
four 1000—mL aliquots of reagent water. A representative wastewater may be used in
place of the reagent water, but one or more additional aliquots must be analyzed to
determine background levels, and the spike level must exceed twice the background level
for the test to be valid. Analyze the aliquots according to the method beginning in
Section 10.
8.2.3 Calculate the average percentage recovery (R), and the standard deviation of the
percentage recovery (s), for the results. Wastewater background corrections must be
made before R and a calculations are performed.
8.2.4 Using the appropriate data from Table 2, determine the recovery and single-operator
precision expected for the method, and compare these results to the values measured in
Section 8.2.3. If the data are not comparable, the analyst must review potential problem
areas and repeat the test.
8.3 The analyst must calculate method performance criteria and define the performance of the
laboratory for each spike concentration and parameter being measured.
8.3.1 Calculate upper and lower control limits for method performance as follows:
Upper Control Limit (UCL) = R + 3s
Lower Control Limit (LCL) = R - 3s
where R and s are calculated as in Section 8.2.3. The UCL and LCL can be used to
construct control tharts that are useful in observing trends in performance.
444
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Method 635
8.3.2 The laboratory must develop and maintain separate accuracy statements of laboratory
performance for wastewater samples. An accuracy statement for the method is defined
as R ± s. The accuracy statement should be developed by the analysis of four aliquots
of wastewater as described in Section 8.2.2, followed by the calculation of R and s.
Alternatively, the analyst may use four wastewater data points gathered through the
requirement for continuing quality control in Section 8.4. The accuracy statements
should be updated with this method. This ability is established as described regularly. 6
8.4 The laboratory is required to collect in duplicate a portion of their samples to monitor spike
recoveries. The frequency of spiked sample analysis must be at least 10% of all samples or one
sample per month, whichever is greater. One aliquot of the sample must be spiked and analyzed
as described in Section 8.2. If the recovery for a particular compound does not fall within the
control limits for method performance, the results reported for that compound in all samples
processed as part of the same set must be qualified as described in Section 13.3. The laboratory
should monitor the frequency of data so qualified to ensure that it remains at or below 5%.
8.5 Before processing any samples, the analyst should demonstrate through the analysis of a 1-L
aliquot of reagent water that all glassware and reagent interferences are under control. Each time
a set of samples is extracted or there is a change in reagents, a laboratory reagent blank should
be processed as a safeguard against laboratory cofltamination.
8.6 It is recommended that the laboratory adopt additional quality assurance practices for use with this
method. The specific practices that are most productive depend upon the needs of the laboratory
and the nature of the samples. Field duplicates may be analyzed to monitor the precision of the
sampling technique. When doubt exists over the identification of a peak on the chromatogram,
confirmatory techniques, such as liquid chromatography with a dissimilar column, must be used.
Whenever possible, the laboratory should perform analysis of standard reference materials and
participate in relevant performance evaluation studies.
9. SAMPLES COLLECTION, PRESERVATION, AND HANDLING
9.1 Grab samples must be collected in glass containers. Conventional sampling practices 7 should be
followed; however, the bottle must not be prerinsed with sample before collection. Composite
samples should be collected in refrigerated glass containers in accordance with the requirements
of the program. Automatic sampling equipment must be as free as possible of Tygon and other
potential sources of contamination.
9.2 The samples must be iced or refrigerated at 4°C from the time of collection until extraction.
9.3 Adjust the pH of the sample to 6 to 8 with iN sodium hydroxide or iN sulfuric acid immediately
after sampling.
10. SAMPLE EXTRACTION
10.1 Mark the water meniscus on the side of the sample bottle for later determination of sample
volume. Pour the entire sample into a 2-L separatory funnel. Check the pH of the sample with
wide range pH paper and adjust to 7 with iN sodium hydroxide or iN H 2 S0 4 .
10.2 Add 60 mL of methylene chloride to the sample bottle, seal, and shake 30 seconds to rinse the
inner walls. Transfer the solvent to the separatory funnel and extract the sample by shaking the
445
-------
Method 635
funnel for 2 minutes with periodic venting to release excess pressure. Allow the organic layer to
separate from the water phase for a minimum of 10 minutes. If the emulsion interface bdween
layers is more than one-third the volume of the solvent layer, the analyst must employ mechanical
techniques to complete the phase separation. The optimum technique depends upon the sample,
but may include stirring, filtration of the emulsion through glass wool, centrifugation, or other
physical methods. Collect the methylene chloride extract in a 250-mL Erlenmeyer flask.
10.3 Add a second 60-mL volume of methylene chloride to the sample bottle and repeat the extraction
procedure a second time, collecting the extract. Perform a third extraction in the same manner
and collect the extract.
10.4 Assemble a Kuderna-Danish (K-D) concentrator by attaching a 25-mL concentrator tube to a
250-mL evaporative flask. Other concentration devices or techniques may be used in place of the
K-D if the requirements of Section 8.2 are met.
10.5 Pour the combined extract through a drying column containing about 10 cm of anhydrous sodium
sulfate, and collect the extract in the K-I) concentrator. Rinse the Erlenmeyer flask and column
with 20 to 30 mL of methylene chloride to complete the quantitative transfer. Once the flask rinse
has passed through the drying column, rinse the column with an additional 30 to 40 mL of
methylene chloride.
10.6 Add one or two clean boiling chips to the evaporative flask and attach a three-ball Snyder column.
Prewet the Snyder column by adding about 1 mL methylene chloride to the top. Place the K-D
apparatus on a hot water bath, 60 to 65°C, so that the concentrator tube is partially immersed in
the hot water, and the entire lower rounded surface of the flask is bathed with hot vapor. Adjust
the vertical position of the apparatus and the water temperature as required to complete the
concentration in 15 to 20 minutes. At the proper rate of distillation, the balls of the column will
actively chatter but the chambers will not flood with condensed solvent. When the apparent
volume of liquid reaches 1 mL, remove the K-D apparatus and allow it to drain and cool for at
least 10 minutes.
10.7 Remove the macro-Snyder column and rinse the flask and its lower joint into the concentrator tube
with 1 to 2 mL of methylene chloride. A 5-mL syringe is recommended for this operation. Add
one or two clean boiling chips and attach a two-ball micro-Snyder column to the concentrator
tube. Prewet the micro-Snyder column with methylene chloride and concentrate the solvent
extract as before. When an apparent volume of 0.5 mL is reached, or the solution stops boiling,
remove the K-D apparatus and allow it to drain and cool fQr 10 minutes. If analysis is being
performed using Column 1 or if s mple cleanup is required, proceed with Section 10.9. If
Column 2 is used and no sample cleanup is required, proceed with Section 10.8.
10.8 Add 10 mL of acetonitrile to the concentrator tube along with one or two clean boiling chips.
Attach a two-ball micro-Snyder column to the concentrator tube. Prewet the micro-Snyder column
with acetonitrile and concentrate the solvent extract as before. When an apparent volume of 1 mL
is reached, remove the K-D apparatus and allow it to drain and cool for 10 minutes. Transfer the
liquid to a 10-mL volumetric flask and dilute to the mark with acetonitrile. Mix thoroughly prior
to analysis. Proceed with Section 12 using Column 2.
10.9 Remove the micro-Snyder column and adjust the volume of the extract to 1.0 mL with methylene
chloride. Stopper the concentrator tube and store refrigerated if further processing will not be
performed immediately. If the extract is to be stored longer than two days, transfer the extract
to a screw-capped vial with a PTFE-lined cap. If the sample extract requires no further cleanup,
446
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Method 635
proceed with the liquid chromatographic analysis in Section 12 using Column 1. If the sample
requires cleanup, proceed to Section 11.
10.10 Determine the original sample volume by refilling the sample bottle to the mark and transferring
the water to a 1000-mL graduated cylinder. Record the sample volume to the nearest 5 mL.
11. CLEANUP AND SEPARA liON
11.1 Cleanup procedures may not be necessary for a relatively clean sample matrix. The cleanup
procedure recommended in this method has been used for the analysis of various clean waters and
industrial effluents. If particular circumstances demand the use of additional cleanup, the analyst
must demonstrate that the recovery of each compound of interest is no less than 85%.
11.2 Slurry l0gofsiicagelin50mLofacetonetowhichhasbeenadded600pLofreagentwater.
Transfer the slurry to a chromatographic column (silica gel is retained with a plug of glass wool).
Wash the column with 100 mL of methylene chloride. Use a column flow rate of 2 to
2.5 mL/min throughout the wash and elution profiles.
11.3 Add the extract from Section 10.9 to the head of the column. Allow the solvent to elute from the
column until the silica gel is almost exposed to the air. Elute the column with 50 mL of
methylene chloride. Discard this fraction.
11.4 Elute the column with 60 mL of 6% acetone in methylene chloride. Collect this fraction in a K-D
apparatus. Concentrate the column fraction to 1 mL as described in Sections 10.6 and 10.7. If
Column 1 is being used, proceed with Section 11.5. If Column 2 is being used, proceed with
Section 11.7.
11.5 Add 5 mL of hexane to the concentrate along with one or two clean boiling chips. Attach a
three-ball micro-Snyder column to the concentrator tube. Prewet the micro-Snyder column with
hexane and concentrate the solvent extract to an apparent volume of 1 mL. Allow the K-D
apparatus to drain and cool for 10 minutes.
11.6 Transfer the liquid to a 10-mL volumetric flask and dilute to the mark with hexane. Mix
thoroughly prior to analysis. If the extracts will not be analyzed immediately, they should be
transferred to PTFE-sealed screw-cap vials and refrigerated. Proceed with the liquid
chromatographic analysis using Column 1.
11.7 Add 10 mL of acetonitrile to the concentrate along with one or two clean boiling chips. Attach
a three-ball micro-Snyder column to the concentrator tube. Prewet the micro-Snyder column with
acetonitrile and concentrate the solvent extract to an apparent volume of 1 mL. Allow the K-D
apparatus to drain and cool for 10 minutes.
11.8 Transfer the liquid to a 10-mL volumetric flask and dilute to the mark with acetonitrile. Mix
thoroughly prior to analysis. If the extracts will not be analyzed immediately, they should be
transferred to PTFE-sealed screw-cap vials and refrigerated. Proceed with the liquid
chromatographic analysis using Column 2.
12. LIQWD CHROMATOGRAPHY
12.1 Table 1 summarizes the recommended operating conditions for the liquid chromatograph.
Included in this table are estimated retention times and method detection limits that can be
achieved by this method. An example of the separations achieved by Column 1 and Column 2
447
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Method 635
are shown in Figures 1 and 2. Other column c, chromatographic conditions, or detectors may be
used if the requirements of Section 8.2 are met.
12.2 Calibrate the liquid chroinatographic system daily as described in Section 7.
12.3 If an internal standard approach is being used, the analyst must not add the internal standard to
sample extracts until immediately before injection into the instrument. Mix thoroughly.
12.4 Inject 5 to 20 1 iL of the sample extract by completely filling the sample valve loop. Record the
resulting peak sizes in area or peak height units.
12.5 The width of the retention-time window used to make identifications should be based upon
measurements of actual retention-time variations of standards over the course of a day. Three
times the standard deviation of a retention time for a compound can be used to calculate a
suggested window size; however, the experience of the analyst should weigh heavily in the
• interpretation of chromatograms.
12.6 If the response for the peak exceeds the working range of the system, dilute the extract and
reanalyze.
12.7 If the measurement of the peak response is prevented by the presence of interferences, further
cleanup is required.
13. CALCULATIONS
13.1 Determine the concentration of individual compounds in the sample.
13.1.1 If the external standard calibration procedure is used, calculate the amount of material
injected from the peak response using the calibration curve or calibration factor in
Section 7.2.2. The concentration in the sample can be calculated as follows:
Equation 2
( A)(V )
Concentration, gJL = (V)(V)
where
A = Amount of material injected, in ng
V = Volume of extract injected, in pL
V 1 = Volume of total extract, in buL
1’, = Volume of wter extracted, in niL
13.1.2 If the internal standard calibration procedure was used, calculate the concentration in the
sample using the response factor (RF) determined in Section 7.3.2 as follows:
448
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Method 635
Equation 3
Concentration, igIL = ( A,)(!, )
(A ,)(RF)(V 0 )
where
A, = Response for parameter to be measured
Ab Response for the internal standard
I , Amount of internal standard added to each extract, in g
= Volume of water extracted, in L
13.2 Report results in micrograms per liter without correction for recovery data. When duplicate and
spiked samples are analyzed, report all data obtained with the sample results.
13.3 For samples processed as part of a set where the laboratory spiked sample recovery falls outside
of the control limits in Section 8.3, data for the affected compounds must be labeled as suspect.
14. METHOD PERFORMANCE
14.1 The method detection limit (MDL) is defined as the minimum concentration of a substance that
can be measured and reported with 99% confidence that the value is about zero. The MDL
concentrations listed in Table 1 were obtained using reagent water. 1 Similar results were achieved
using representative wastewaters.
14.2 This method has been tested for linearity of recovery from spiked reagent water and has been
demonstrated to be applicable over the concentration range from 10 X MDL to 1000 x MDL.
14.3 In a single laboratory, Battelle Columbus Laboratories, using spiked wastewater samples, the
average recoveries presented in Table 2 were obtained. Seven replicates of each of two different
wastewaters were spiked and analyzed. The standard deviation of the percent recovery is also
included in Table 2.’
449
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Method 635
References
1. “Development of Methods for Pesticides in Wastewaters,” Report for EPA Contract 68-03-2956
(In preparation).
2. ASTM Annual Book of Standards, Part 31, D3694, “Standard Practice for Preparation of Sample
Containers and for Preservation,” American Society for Testing and Materials, Philadelphia,
Pennsylvania, p. 679, 1980.
3. “Carcinogens - Working with Carcinogens,” Department of Health, Education, and Welfare,
Public Health Service, Center for Disease Control, National Institute for Occupational Safety and
Health, Publication No. 77-206, August, 1977.
4. “OS}IA Safety and Health Standards, General Industry,” (29 CFR 1910), Occupational Safety and
Health Administration, OSHA 2206 (Revised, January, 1976).
5. “Safety in Academic Chemistry Laboratories,” American Chemical Society Publication,
Committee on Chemical Safety, 3rd Edition, 1979.
6. “Handbook for Analytical Quality Control in Water and Wastewater Laboratories,”
EPA.600/4-79-019, U. S. Environmental Protection Agency, Environmental Monitoring and
Support Laboratory - Cincinnati, Ohio, March, 1979.
7. ASTM Annual Book of Standards, Part 31, D3370, “Standard Practice for Sampling Water,”
American Society for Testing and Materials, Pluladelphia, Pennsylvania, p 76, 1980
8. Glaser, J. A. et al, “Trace Analysis for Wastewaters,” Environmental Science and Technology,
15, 1426 (1981).
450
-------
Method 635
Table 1. Chromatographic Conditions and Method Detection Limits
Retention Time (mm.,) Method Detection
Limit
Parameter Column 1 Column 2 (pg/LI
Rotenone 8.6 8.0 1.6
Rotenone
Column 1 conditions: Zorbax-CN, 5 micron, 250 by 4.6 mm; 1 mL/min flow; 30/70
methylene chioride/hexane.
Column 2 conditions: Spherisorb-ODS, 5 micron, 250 by 4.6 mm; 1 mL/min flow;
60/40 acetonitrile/water.
Table 2. Single-Laboratory Accuracy and Precision (a)
Parameter Average Standard Spike Level Number of Matrix
Percent Deviation (ugiL) Analyses Type (b)
Recovery Percent
Rotenone 85 8 5.5 7 1
88 3 109 7 1
(a) Column 1 conditions were used.
(b) 1 = Pesticide manufacturing wastewater
457
-------
M9Uwd 5
‘I
Rotenone
/
I I I I I I I I I I I I I I I I
0 1.2 2.4 3.0 4.8 6.0 7.2 8.4 9.0 10.0 12.0
Retention Time (minutes)
52 OO2 25
Figure 1. HPLC-UV Chromatogram of Standard Solution Representing 5 p.g/L
of Rotenone in Water (Column 1).
452
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Method 635
Figure 2. HPLC-UV Chromatogram of Standard Solution Representing 200 pgIL
of Rotenone in Water (Column 2.)
Rotenone
/
4.5 6.0
10.5
A -OO22B
I I I I I
12.0 13.5 15.0
453
-------
Method 636
The Determination of Bensulide
in Municipal and Industrial
Waste water
-------
Method 636
The Determination of Bensulide in Municipal and Industrial
Wastewater
1Sc0PE AND APPLICATION
1 .1 This method covers the determination of bensulide pesticide. The following parameter can be
determined by this method:
Parameters CAS No.
Bensulide 74 1-58-2
1.2 This is a high performance liquid chromatographic (HPLC) method applicable to the determination
of the compound listed above in municipal and industrial discharges as provided under 40 CFR
136.1. Any modification of this method beyond those expressly permitted shall be considered a
major modification subject to application and approval of alternative test procedures under .40 CFR
136.4 and 136.5.
1.3 The method detection limit (MDL, defined in Section 14) for bensulide compound is listed in
Table 1. The MDL for a specific wastewater may differ from those listed, depending upon the
nature of interferences in the sample matrix.
1.4 This method is restricted to use by or under the supervision of analysts experienced in the use of
liquid chromatography and in the interpretation of liquid chromatograms. Each analyst must
demonstrate the ability to generate acceptable results with this method using the procedure
described in Section 8.2.
1.5 When this method is used to analyze unfamiliar samples for the compound above, compound
identifications should be supported by at least one additional qualitative technique. This method
describes analytical conditions for a second liquid chromatographic column that can be used to
confirm measurements made with the primary column.
2. SUMMARY OF METHOD
2.1 A measured volume of sample, approximately 1 L, is extracted with methylene chloride using a
separatory funnel. The methylene chloride extract is dried and exchanged to acetonitrile during
concentration to a volume of 2 mL or less. Liquid chromatographic conditions are described
which permit the separation and measurement of the compounds in the extract by HPLC-UV.’
3. INTERFERENCES
3.1 Method interferences may be caused by contaminants in solvents, reagents, glassware, and other
sample processing hardware that lead to discrete artifacts or elevated baselines in liquid
chromatograms. All of these materials must be routinely demonstrated to be free from
interferences under the conditions of the analysis by running laboratory reagent blanks as
described in Section 8.5.
457
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Method 636
3.1.1 Glassware must be scrupulously cleaned. 2 Clean all glassware as soon as possible after
use by thoroughly rinsing with the last solvent used in it. Follow by washing with hot
water and detergent and thorough rinsing with tap and reagent water. Drain dry, and
heat in an oven or muffle furnace at 400°C for 15 to 30 minutes. Some thermally stable
materials, such as PCBs, may not be eliminated by this treatment. Thorough rinsing with
acetone and pesticide-quality hexane may be substituted for the heating. After drying and
cooling, seal and store glassware in a clean environment to prevent any accumulation of
dust or other confaui1nant . Store inverted or capped with aluminum foil.
3.1.2 The use of high-purity reagents and solvents helps to miniimize interference problems.
Purification of solvents by distillation in all-glass systems may be required.
3.2 Matrix interferences may be caused by contaminants that are coextracted from the sample. The
extent of matrix interferences will vary considerably from source to source, depending upon the
n uIe and diversity of the industrial complex or municipality being sampled. The cleanup
procedure in Section 11 can be used to overcome these interferences, but unique samples may
require additional cleanup approaches to achieve the MDL listed in Table 1.
4. SAFETY
4.1 The toxicity or carcinogenicity o each reagent used in this method has not been precisely defined;
however, each chemical compound should be treated as a potential health hazard. From this
viewpoint, exposure to these chemicals must be reduced to the lowest possible level by whatever
, avallable The laboratory is responsible for maintaining a current awareness file of OSHA
regulations regarding the safe haMling of the chemicals specified in this method. A reference file
of material data handling sheets should also be made available to all personnel involved in the
chemical analysis. Additional references to laboratory safety are available and have been
ideiuifled” for the information of the analyst.
5. Apw injs AND MA TERJALS
5.1 Sampling equipment, for discrete or composite sampling.
5.1.1 Grab-sample bottle: Borosilicate or flint glass, 1-L or 1-quart volume, fitted with screw-
caps lined with P’WB . Aluminum foil may be substituted for PTFE if the sample is not
corrosive. The container and cap liner must be washed, rinsed with acetone or methylene
chloride, and dried before use to minimize contamination.
5.1.2 Autematic sampler (optional): Must incorporate glass sample containers for the
collection of a minimum of 250 mL. Sample containers must be kept refrigerated at 4°C
and protected from light during compositing. If the sampler uses a peristaltic pump, a
minimum length of compressible silicone rubber tubing may be used. Before use,
however, the compressible tubing should be thoroughly rinsed with methanol, followed
by repeated rinsings with distilled water to minimize the potential for contamination of
the sample. An integrating flow meter is required to collect flow-proportional
composites.
5.2 Glassware. (All specifications are suggested. Catalog numbers are included for illustration only.)
5.2.1 Separatory funnel: 2000-mL, with PTFE stopcock.
458
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Method 636
5.2.2 Drying column: Chromatographic column 400 mm long by 10 mm ID with coarse frit.
5.2.3 Chromatographic column: 400 mm long by 19 mm II) with 250-mL reservoir at the top
and PTFE stopcock (Kontes K-420290 or equivalent).
5.2.4 Concentrator tube, Kuderna-Danish: 25-mL, graduated (Kontes K-570050-2525 or
equivalent). Calibration must be checked at the volumes employed in the test. A
ground-glass stopper is used to prevent evaporation of extracts.
5.2.5 Evaporative flask, Kuderna-Danish: 500-mL (Kontes K-570001-0500 or equivalent).
Attach to concentrator tube with springs.
5.2.6 Snyder column, Kuderna-Danish: Three-ball macro (Kontes K-503000-0121 or
equivalent).
5.2.7 Snyder column, Kuderna-Danish: Two-ball micro (Kontes K-569001-0219 or
equivalent).
5.2.8 Vials: Amber glass, 5- to 10-mL capacity with PTFE-lined screw-cap.
5.3 Boiling chips: Approximately 10/40 mesh carborundum. Heat to 400°C for 4 hours or extract
in a Soxhiet extractor with methylene chloride.
5.4 Water bath: Heated, capable of temperature control (±2°C). The bath should be used in a hood.
5.5 Balance: Analytical, capable of accurately weighing to the nearest 0.0001 g.
5.6 Liquid chromatograph: Analytical system complete with liquid chromatograph and all required
accessories including syringes, analytical columns, detector and strip-chart recorder. A data
system is recommended for measuring peak areas.
5.6.1 Pump: Isocratic pumping system, constant flow.
5.6.2 Column 1: Reversed-phase column, 5 i Spherisorb-ODS, 250 mm long by 4.6 mm ID
or equivalent.
5.6.3 Column 2: Reversed-phase column, 5 Lichrosorb RP-2, 250 mm long by 4.6 mm ID
or equivalent.
5.6.4 Detector: Ultraviolet absorbance detector, 270 mm.
6. REAGENTs
6.1 Reagent water: Reagent water is defined as a water in which an interferent is not observed at the
method detection limit of each parameter of interest.
6.2 Methylene chloride, methanol, acetonitrile: Distilled-in-glass quality or equivalent.
6.3 Sodium sulfate: ACS, granular, anhydrous; heated in a muffle furnace at 400°C overnight.
6.4 Sodium phosphate, monobasic: ACS, crystal.
6.5 iN sulfuric acid: Slowly add 2.8 mL of concentrated H 2 S0 4 (94%) to about 50 mL of distilled
water. Dilute to 100 mL with distilled water.
6.6 iN sodium hydroxide: Dissolve 4.0 grams of NaOH in 100 mL of distilled water.
6.7 Florisil: PR grade (601100 mesh). Purchase activated at 675 °C and store in brown glass bottle.
To prepare for use, place 150 g in a wide-mouth jar and heat overnight at 160 to 170°C. Seal
tightly with PTFE or aluminum foil-lined screw-cap and cool to room temperature.
459
-------
Method 636
6.8 Stock standard solutions (1.00 gIpl): Stock standard solutions can be prepared from pure
standard materials or purchased as certified solutions.
6.8.1 Prepare stock standard solutions by accurately weighing about 0.0100 g of pure material.
Dissolve the material in distilled-in-glass quality methanol and dilute to volume in a
l0-mL volumetric flask. Larger volumes can be used at the convenience of the analyst.
If compound purity is certified at 96% or greater, the weight can be used without
correction to calculate the concentration of the stock standard. Commercially prepared
stock standards can be used at any concentration if they are certified by the manufacturer
or by an independent source.
6.8.2 Transfer the stock standard solutions into PTFE-sealed screw-cap bottles. Store at 4°C
and protect from light. Stock standard solutions should be checked frequently for signs
of degradation or evaporation, especially just prior to preparing calibration standards
from them.
6.8.3 Stock standard solutions must be replaced after 6 months, or sooner if comparison with
check standards indicates a problem.
7. CALL8RAT1ON
7.1 Establish liquid chromaiographic operating parameters equivalent to those indicated in Table 1.
The liquid chromatographic system can be calibrated using the external standard technique (Section
7.2) or the internal standard technique (Section 7.3).
7.2 External standard calibration procedure:
7.2.1 For each compound of interest, prepare calibration standards at a minimum of three
concentration levels by adding volumes of one or more stock standards to a volumetric
flask and diluting to volume with acetonitrile. One of the externaL standards should be
at a concentration near, but above, the method detection limit. The other concentrations
should correspond to the expected range of concentrations found in real samples or
should define the working range of the detector.
7.2.2 Using injections of 2 to 5 pL of each calibration standard, tabulate peak height or area
responses against the m -t injected. The results can be used to prepare a calibration
curve for each compound. Alternatively, the ratio of the response to the mass injected,
defined as the calibration factor (CF), can be calculated for each compound at each
standard concentration. If the relative standard deviation of the calibration factor is less
than 10% over the working range, linearity through the origin can be assumed and the
average calibration factor can be used in place of a calibration curve.
7.2.3 The working calibration curve or calibration factor must be verified on each working
shift by the measurement of one or more calibration standards. If the response for any
compound varies from the predicted response by more than ± 10%, the test must be
repeated using a fresh calibration standard. Alternatively, a new calibration curve or
calibration factor must be prepared for that compound.
7.3 Internal standard calibration procedure: To use this approach, the analyst must select one or more
internal standards similar in analytical behavior to the compounds of interest. The analyst must
further demonstrate that the measurement of the internal standard is not affected by method or
460
-------
Method 636
matrix interferences. Due to these limitations, no internal standard applicable to all samples can
be suggested.
7.3.1 prepare calibration standards at a minimum of three concentration levels for each
compound of interest by adding volumes of one or more stock standards to a volumetric
flask. To each caiibration standard, add a known constant amount of one or more
internal standards, and dilute to volume with acetonitrile. One of the standards should
be at a concentration near, but above, the method detection limit. The other
concentrations should correspond to the expected range of concentrations found in real
samples, or should define the working range of the detector.
7.3.2 Using injections of 2 to 5 ,LL of each calibration standard, tabulate the peak height or
area responses against the concentration for each compound and internal standard.
Calculate response factors (RF) for each compound as follows:
Equation 1
= ( A,C , )
(ARC)
where
A, = Response for the compound to be measured
A,, = Response for the internal standard
C,, = Concentration of the intermaistandard, in g/L
C, = Concentration of the compound to be measured, in 1 igIL
If the RF value over the working range is constant, less than 10% relative standard
deviation, the RF can be assumed to be invariant and the average RF can be used for
calculations. Alternatively, the results can be used to plot a calibration curve of response
ratios, A,/A 1 against RF.
7.3.3 The working calibration curve or RF must be verified on each working shift by the
measurement of one or more calibration standards. If the response for any compound
varies from the predicted response by more than ±10%, the test must be repeated using
a fresh calibration standard. Alternatively, a new calibration curve must be prepared for
that compound.
7.4 Before using any cleanup procedure, the analyst must process a series of calibration standards
through the procedure to validate elution patterns and the absence of interferences from the
reagents.
8. QuAun CONTROL
8.1 Each laboratory using this method is required to operate a formal quality control program. The
minimum requirements of this program consist of an initial demonstration of laboratory capability
and the analysis of spiked samples as a continuing check on performance. The laboratory is
required to maintain performance records to define the quality of data that is generated.
461
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Method 636
8.1.1 Before performing any analyses, the analyst must demonstrate the ability to generate
acceptable accuracy and precision with this method. This ability is established as
described in Section 8.2.
8.1.2 In recognition of the rapid advances occurring in chromatography, the analyst is
permitted certain options to improve the separations or lower the cost of measurements.
Each time such modifications to the method are made, the analyst is required to repeat
the procedure in Section 8.2.
8.1.3 The laboratory must spike and analyze a minimum of 10% of all samples to monitor
continuing laboratory performance. This procedure is described in Section 8.4.
8.2 To establish the ability to generate acceptable accuracy and precision, the analyst must perform
the following operations.
8.2.1 Select a representative spike concentration for each compound to be measured. Using
stock standards, prepare a quality control check sample concentrate in methanol 1000
times more concentrated than the selected concentrations.
8.2.2 Using a pipette, add 1.00 mL of the check sample of each of a minimum of four
1000-mL aliquots of reagent water. A representative wastewater may be used in place
of the reagent water, but one or more additional aliquots must be analyzed to determine
background levels, and the spike level must exceed twice the background level for the
test to be valid. Analyze the aliquots according to the method beginning in Section 10.
8.2.3 Calculate the average percent recovery (R), and the standard deviation of the percent
recovery (s), for the results. Wastewater background corrections must be made before
R and s calculations are performed.
8.2.4 Using the appropriate data from Table 2, determine the recovery and single-operator
precision expected for the method, and compare these results to the values measured in
Section 8.2.3. If the data are not comparable, the analyst must review potential problem
areas and repeat the test.
8.3 The analyst must calculate method performance criteria and define the performance of the
laboratory for each spike concentration and parameter being measured.
8.3.1 Calculate upper and lower control limits for method performance as follows:
Upper Control Limit (UCL) = R + 3s
Lower Control Limit (LCL) = R -3s
where R and s are calculated as in Section 8.2.3. The UCL and LCL can be used to
construct control charts 6 that are useful in observing trends in performance.
8.3.2 The laboratory must develop and maintain separate accuracy statements of laboratory
performance for wastewater samples. An accuracy statement for the method is defined
as R ± s. The accuracy statement should be developed by the analysis of four aliquots
of wastewater as described in Section 8.2.2, followed by the calculation of R and S.
Alternatively, the analyst may use four wastewater data points gather through the
requirement for continuing quality control in Section 8.4. The accuracy statements
should be updated with this method. This ability is established as described regularly. 6
462
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Method 636
8.4 The laboratory is required to collect in duplicate a portion of their samples to monitor spike
recoveries. The frequency of spiked sample analysis must be at least 10% of all samples or one
sample per month, whichever is greater. One aliquot of the sample must be spiked and analyzed
as described in Section 8.2. If the recovery for a particular compound does not fall within the
control limits for method perfonnance, the results reported for that compound in samples
processed as part of the same set must be qualified as described in Section 13.3. The laboratory
should monitor the frequency of data so qualified to ensure that it remains at or below 5%.
8.5 Before processing any samples, the analyst should demonstrate though the analysis of a 1-L aliquot
of reagent water that all glassware and reagent interferences are under control. Each time a set
of samples is extracted or there is a change in reagents, a laboratory reagent blank should be
processed as a safeguard against laboratory contamination.
8.6 It is recommended that the laboratory adopt additional quality assurance practices for use with this
method. The specific practices that are most productive depend upon the needs of the laboratory
and the nature of the samples. Field duplicates may be analyzed to monitor the precision of the
sampling technique. When doubt exists over the identification of a peak on the chromatogram,
confirmatory techniques such as liquid chromatography with a dissimilar column, must be used.
Whenever possible, the laboratory should perform analysis of standard reference materials and
participate in relevant performance evaluation st idies.
9. SAMPLES COLLECTION, PRESERVATiON, AND HANDUNG
9.1 Grab samples must be collected in glass containers. Conventional sampling practices 1 should be
followed; however, the bottle must not be prerinsed with sample before collection. Composite
samples should be collected in refrigerated glass containers in accordance with the requirements
of the program. Automatic sampling equipment must be as free as possible of Tygon and other
potential sources of contamination.
9.2 The samples must be iced or refrigerated at 4°C from the time of collection until extraction.
9.3 Adjust the pH of the sample to 6 to 8 with iN sodium hydroxide or iN sulfuric acid immediately
after sampling.
10. SAMPLE EXTRACTiON
10.1 Mark the water meniscus on the side of the sample bottle for later determination of sample
volume. Pour the entire sample into a 2-L separatory funnel. Check the pH of the sample with
wide range pH paper and adjust to 7 with iN sodium hydroxide or iN H 2 S0 4 .
10.2 Add 60 mL of methylene chloride to the sample bottle, seal, and shake 30 seconds to rinse the
inner walls. Transfer the solvent to the separatory funnel and extract the sample by shaking the
funnel for two minutes with periodic venting to release excess pressure. Allow the organic layer
to separate from the water phase for a minimum of 10 minutes. If the emulsion interface between
layers is more than one-third the volume of the solvent layer, the analyst must employ mechanical
techniques to complete the phase separation. The optimum technique depends upon the sample,
but may include stirring, filtration of the emulsion through glass wool, centrifugation, or other
physical methods. Collect the methylene chloride extract in a 250-mL Erlenmeyer flask.
463
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Method 636
10.3 Add a second 60-mi.. volume of methylene chloride to the sample bottle and repeat the extraction
procedure a second time, collecting the extract. Perform a third extraction in the same manner
and collect the extract.
10.4 Assemble a Kuderna-Danish (K-D) concentrator by attaching a 25-mi concentrator tube to a
500-mi.. evaporative flask. Other concentration devices or techniques may be used in place of the
K-D if the requirements of Section 8.2 are met.
10.5 Pour the combined extract through a drying column containing about 10 cm of anhydrous sodium
sulfate, and collect the extract in the K-D concentrator. Rinse the Erlenmeyer flask and column
with 20 to 30 mL of methylene chloride to complete the quantitative transfer. Once the flask rinse
has passed through the drying column, rinse the column with 30 to 40 mL of methylene chloride.
10.6 Add one or two clean boiling chips to the evaporative flask and attach a three-ball macro-Snyder
column. Prewet the Snyder column by adding about 1 mL methylene chloride to the top. Place
the K-D apparatus on a hot water bath, 60 to 65°C, so that the concentrator tube is partially
immersed in the hot water, and the entire lower rounded surface of the flask is bathed with hot
vapor. Adjust the vertical position of the apparatus and the water temperature as required to
complete the concentration in 15 to 20 minutes. At the proper rate of distillation, the balls of the
column will actively chatter but the chambers will not flood with condensed solvent. When the
apparent volume of liquid reaches 1 mL, remove the K-D apparatus and allow it to drain and cool
for at least ten minutes. If the sample extract requires no further cleanup, proceed with solvent
exchange to acetonitrile and chromatographic analysis as described in Sections 11.5 and 12
respectively. If the sample requires cleanup, proceed to Section 10.7.
10.7 Remove the macro-Snyder column and rinse the flask and its lower joint into the concentrator tube
with 1 to 2 mL of methylene chloride. A 5-mi syringe is recommended for this operation. Add
one or two clean boiling chips and attach a two-ball micro-Snyder column to the concentrator
tube. Prewet the micro-Snyder column with methylene chloride and concentrate the solvent
extract as before. When an apparent volume of 0.5 mL is reached, or the solution stops boiling
remove the K-D apparatus and allow it to drain and cool for 10 minutes.
10.8 Remove the micro-Snyder column and adjust the volume of the extract to 1.0 mL with methylene
chloride. Stopper the concentrator tube and store refrigerated if further processing will not be
performed immediately. If the extract is to be stored longer than 2 days, transfer the extract to
a screw-capped vial with a PTFE-lined cap.
10.9 Determine the original sample volume by refilling the sample bottle to the mark and transferring
the water to a 1000-mL graduated cylinder. Record the sample volume to the nearest 5 mL.
11. CLEANUP AND SEPARA T1ON
11.1 Cleanup procedures may not be necessary for a relatively clean sample matrix. The cleanup
procedure recommended in this method has been used for the analysis of various clean waters and
industrial effluents. If particular circumstances demand the use of additional cleanup, the analyst
must demonstrate that the recovery of each compound of interest is no less than 76%.
11.2 Slurry 10 g of Florisil in 100 mL of methylene chloride which has been saturated with reagent
water. Transfer the slurry to a chromatographic column (Florisil may be retained with a plug of
glass wool). Wash the column with 100 mL of methylene chloride. Use a column flow rate of
2 to 2.5 mL/min throughout the wash and elution profiles.
464
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Method 636
11.3 Add the extract from Section 10.8 to the head of the column. Allow the solvent to elute from the
column until the Florisil is almost exposed to the air. Elute the column with 50 mL of methylene
chloride. Discard this fraction.
11.4 Elute the column with 50 mL of 5% acetone in methylene chloride. Collect this fraction in a K-D
apparatus. Concentrate the column fraction to 1 mL as described in Sections 10.6 and 10.7.
11.5 Add 15 mL of acetonitrile to the concentrate along with one or two clean boiling chips. Attach
a three-ball micro-Snyder column to the concentrator tube. Prewet the micro-Snyder column with
acetonitrile and concentrate the solvent extract to an apparent volume of 1 mL. Allow the K-D
apparatus to drain and cool for 10 minutes.
11.6 Transfer the liquid to a 2-mL volumetric flask and dilute to the mark with acetomtrile. Mix
thoroughly prior to analysis. If the extracts will not be analyzed immediately, they should be
transferred to PTFE-sealed screw-cap vials and refrigerated. Proceed with the liquid
chromatographic analysis.
12. LIQUID CHROMATOGRAPHY
12.1 Table 1 summarizes the recommended operating conditions for the liquid chromatograph.
Included in this table are estimated retention times and method detection limits that can be
achieved by this method. An example of the separations achieved by Column 1 and Column 2
are shown in Figure 1. Other HPLC columns, chromatographic conditions, or detectors may be
used if the requirements of Section 8.2 are met.
12.2 Calibrate the liquid chromatographic system daily as described in Section 7.
12.3 If an internal standard approach is being used, the analyst must not add the internal standard to
the sample extracts until immediately before injections into the instrument. Mix thoroughly.
12.4 Inject 2 to 5 1 iL of the sample extract into the sample valve loop. Record the resulting peak sizes
in area or peak heights units.
12.5 The width of the retention-time window used to make identifications should be based upon
measurements of actual retention-time variations of standards over the course of a day. Three
times the standard deviation of a retention time for a compound can be used to calculate a
suggested window size; however, the experience of the analyst should weigh heavily in the
interpretation of chromatograms.
12.6 If the response for the peak exceeds the working range of the system, dilute the extract and
reanalyze.
12.7 If the measurement of the peak response is prevented by the presence of interferences, further
cleanup is required.
13. CALCULA TJONS
13.1 Determine the concentration of individual compounds in the sample.
13.1.1 If the external standard calibration procedure is used, calculate the amount of material
injected from the peak response using the calibration curve or calibration factor in
Section 7.2.2. The concentration in the sample can be calculated as follows:
465
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Method 636
Equation 2
Concentration, g/L =_____
where
A = Amount of material injected, in nanograms
V, = Volume of extract injected, in gIL
Vt = Volume of total extract, in g/L
V, = Voiwne of w2er extracted, in mL
13.1.2 If the internal standard calibration procedure was used, calculate the concentration in the
sample using the response factor (RF) determined in Section 7.3.2 as follows:
Equation 3
Concentration, gIL (A ,)(RF)(V ,)
where
A, =
4, =
I, =
V 0 =
Response for parameter to be measured
Response for the internal standard
Amount of internal standard added to each
Volume of water extracted, in liters
extract,
in
g
13.2 Report results in microgram per liter without correction for recovery data. When duplicate and
spiked samples are analyzed, report all data obtained with the sample results.
13.3 For samples processed as part of a set where the laboratory spiked sample recovery falls outside
of the control limits in Section 8.3, data for the affected compounds must be labeled as suspect.
14. METHOD PERFORMANCE
14.1 The method detection limit (MDL) is defined as the minimum concentration of a substance that
can be measured and reported with 99% confidence that the value is above zero.’ The MDL
concentrations listed in Table 1 were obtained using reagent water.’ Similar results were achieved
using representative wastewaters.
14.2 This method has been tested for linearity of recovery from spiked reagent water and has been
demonstrated to be applicable over the concentration range from 10 x MDL to 1000 X MDL.
14.3 In a single laboratory, Battelle Columbus Laboratories, using spiked wastewater samples, the
average recoveries presented in Table 2 were obtained. Seven replicates of each of two different
wastewaters were spiked and analyzed. The standard deviation of the percent recovery is also
included in Table 2.1
466
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Method 636
References
1. “Development of Methods for Pesticides in Wastewaters,” EPA Contract Report 68-03-2956 (In
preparation).
2. ASTM Annual Book of Standards, Part 31, D3694, “Standard Practice for Preparation of Sample
Containers and for Preservation,” American Society for Testing and Materials, Philadelphia,
Pennsylvania, p. 679, 1980.
3. “Carcinogens - Working with Carcinogens,” Department of Health, Education, and Welfare,
Public Health Service, Center for Disease Control, National Institute for Occupational Safety and
Health, Publication No. 77-206, August, 1977.
4. “OSHA Safety and Health Standards, General Industry,” (29 CFR 1910), Occupational Safety and
Health Administration, OSHA 2206 (Revised, January, 1976).
5. “Safety in Academic Chemistry Laboratories,” American Chemical Society Publication,
Committee on Chemical Safety, 3rd Edition, 1979.
6. “Handbook for Analytical Quality Control in Water and Wastewater Laboratories,”
EPA-600/4-79-019, U. S. Environmental Protection Agency, Environmental Monitoring and
Support Laboratory - Cincinnati, Ohio, March, 1979.
7. ASTM Annual Book of Standards, Part 31, 03370, “Standard Practice for Sampling Water,”
American Society for Testing and Materials, Philadelphia, Pennsylvania, p. 76, 1980.
8. Glaser, J. A. et a!, “Trace Analysis for Wastewaters , “ Environmental Science and Technology,
15, 1426, (1981).
467
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Method 636
Table 1. Chromatographic Conditions and Method Detection Limits
Parameter Retention Time (ml,,. ) Method Detection Limit
Column 1 Column 2 (ugiL)
Bensulide 14.1 7.2 1.6
Column 1 conditions: Spherisorb ODS, 5 i, 250 mm long by 4.6 mm ID; 1 mL/min flow; 55/45
acetonitrile/
Column 2 conditions: Lichrosorb RP-2, 5 , 250 mm long by 4.6 mm ID; 1 mL/min flow; 60/40
acetonitrile/water.
Table 2. Single-Laboratory Accuracy and Precision(’)
Average Standard Spike Number
Percent Deviation, Lelve of Matrix
Parameter Recovery (ugiL) Analyses Type (b)
Bensulide 86 18 25 7 1
76 18 250 7 1
(a) Column 1 conditions were used.
(b) 1 = Relevant industrial wastewater
468
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Method 636
Retention Time (minutes)
Figure 1. HPLC-UV Chromatogram of 60 ng of Bensulide (Column 1).
*52-002-27
1.5 3.0 4.5
Bensulide
12.0 13.5 15.0
469
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Method 637
The Determination of MB TS
and TCMTB in Municipal and
Industrial Waste waters
-------
Method 637
The Determination of MBTS and TCMTB in Municipal and Industrial
Wastewaters
1. SCOPE AND APPLICA TION
1.1 This method covers the determination of MBTS and TCMTB pesticides. The following
parameters can be determined by this method.
Parameter CAS No.
MBTS 120-78-5
TCMTB 21564-17-0
1.2 This is a liquid chromatographic (LC) method applicable to the determination of the compounds
listed above in municipal and industrial discharges as provided under 40 CFR 136.1. Any
modification of this method beyond those expressly permitted shall be considered a major
modification subject to application and approval of alternative test procedures under 40 CFR 136.4
and 136.5.
1.3 The method detection limit (MDL, defined in Section 14) for each parameter is listed in Table 1.
The MDL for a specific wastewater may differ from those listed, depending upon the nature of
interferences in the sample matrix.
1.4 The sample extraction and concentration steps in this method are essentially the same as in certain
other 600-series methods. Thus, a single sample may be extracted to measure the compounds
included in the scope of the methods. When cleanup is required, the concentration levels must
be high enough to permit selecting aliquots, as necessary, in order to apply appropriate cleanup
procedures.
1.5 This method is restricted to use by or under the supervision of analysts experienced in the use of
liquid chromatography and in the interpretation of liquid chromatograms. Each analyst must
demonstrate the ability to generate acceptable results with this method using the procedure
described in Section 8.2
1.6 When this method is used to analyze unfamiliar samples for any or all of the compounds above,
compound identifications should be supported by at least one additional qualitative technique.
This method describes analytical conditions for a second liquid chromatographic column that can
be used to confirm measurements made with the primary column.
2. SUMMARY OF METHOD
2.1 A measured volume of sample, approximately 1 L, is extracted with methylene chloride using a
separatory funnel. The methylene chloride extract is dried and concentrated to 5.0 mL. Liquid
chromatographic conditions are described which permit the separation and measurement of the
compounds in the extract by high performance liquid chromatography with ultraviolet detection. 1
2.2 This method provides a silica gel column cleanup procedure to aid in the elimination of
interferences which may be encountered.
473
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Method 637
3. INTERFERENCES
3.1 Method interferences may be caused by contaminants in solvents, reagents, glassware, and other
sample-processing apparatus that lead to discrete artifacts or elevated baselines in liquid
chromatograms. All reagents and apparatus must be routinely demonstrated to be free from
interferences under the conditions of the analysis by running laboratory reagent blanks as
described in Section 85.
3.1.1 Glassware must be scrupulously cleaned. 2 Clean all glassware as soon as possible after
use by thoroughly rinsing with the last solvent used in it. Follow by washing with hot
water and detergent and thorough rinsing with tap and reagent water. Drain dry, and
heat in an oven or muffle furnace at 400°C fur 15 to 30 minutes. Do not heat volumetric
ware. Some thermally stable materials, such as PCBs, may not be eliminated by this
treatment. Thorough rinsing with acetone and pesticide-quality hexane may be substituted
fur the heating. After drying and cooling, seal and store glassware in a clean
environment to prevent any accwmilatlon of dust or other cont min2nts . Store inverted
or capped with slu’n ám Ibil.
3.1.2 The use of high -pirity reagents and solvents helps to minimize interference problems.
Purification of solvents by distillation In all-glass systems may be required.
3.2 Matrix interferences may be caused by contaminants that are coextracted from the sample. The
exle 4 of matrix interferences will vary considerably from source to source, depending upon the
‘ fure and diversity of the industrial complex or municipality sampled. The cleanup procedure
in Section 11 can be used to overcome many of these interferences, but unique samples may
require additional cleamip approaches to achieve the MDL listed in Table 1.
4. SA FE V
4.1 The toxicity or carcinogemicity of each reagent used in this method has not been precisely defined;
however, each chemical compound should be treated as a potential health hazard. From this
viewpoint, exposure to these dimnicals must be reduced to the lowest possible level by whatever
means available. The laboratory is responsible for maintaining a current awareness file of OSHA
regulations regarding the safe h*pdling of the chemicals specified in this method. A reference file
of material data hiTvfling sheets should also be made available to all personnel involved in the
chemical analysis. Additional references to laboratory safety are available and have been
ldesnifled’ for the Information of the analyst.
5. APPARA TUS AND MA TEPJALS
5.1 Sampling equipment, for discrete or composite sampling.
51.1 Grab-sample bottle: Amber borosilicate or flint glass, 1-L or 1-quart volume, fitted with
screw-caps lined with i’WE . Foil may be substituted for PTFE if the sample is not
corrosive. If amber bottles are not available, protect samples from light. The container
and cap liner must be washed, rinsed with acetone or methylene chloride, and dried
before use to minimize contamination.
5.1.2 Automatic sampler (optional): Must incorporate glass sample containers for the
collection of a minimum of 250 mL. Sample containers must be kept refrigerated at 4°C
474
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Method 637
and protected from light during compositing. If the sampler uses a peristaltic pump, a
minimum length of compressible silicone rubber tubing may be used. Before use,
however, the compressible tubing should be thoroughly rinsed with methanol, followed
by repeated rinsings with distilled water to minimize the potential for contamination of
the sample. An integrating flow meter is required to collect flow-proportional
composites.
5.2 Glassware. (All specifications are suggested. Catalog numbers are included for illustration only.)
5.2.1 Separatory funnel: 2000-mL, with PTFE stopcock.
5.2.2 Drying column: Chromatographic column 400 mm long by 10 mm II) with coarse fit.
5.2.3 Chromatographic column: 400 mm long by 19 mm ID with 250-mL reservoir at the top
and PTFE stopcock (Kontes K-420290 or equivalent).
5.2.4 Concentrator tube, Kuderna-Danish: 10-mL, graduated (Kontes K-570050-1025 or
equivalent). Calibration must be checked at the volumes employed in the test. A
ground-glass stopper is used to prevent evaporation of extracts.
5.2.5 Evaporative flask, Kuderna-Danish: 500-mL (Kontes K-570001-0500 or equivalent).
Attach to concentrator tube with springs.
5.2.6 Snyder column, Kuderna-Danish: Three-ball macro (Kontes K-503000-0121 or
equivalent).
5.2.7 Vials: Amber glass, 10- to 15-mL capacity with PTFE-lined screw-cap.
5.2.8 Erlenmeyer flask: 250-mL.
5.2.9 Graduated cylinder 1000-mL.
5.2.10 Volumetric flask: 5-mL, 10-mL.
5.3 Boiling chips: Approximately 10/40 mesh carborundum. Heat at 400°C for 4 hours or perform
a Soxhiet extraction with methylene chloride.
5.4 Water bath: Heated, capable of temperature control (±2°C). The bath should be used in a hood.
5.5 Balance: Analytical, capable of accurately weighing to the nearest 0.0001 g.
5.6 Liquid chromatograph: Analytical system complete with liquid chromatograph suitable for
on-column injection and all required accessories including syringes, analytical columns, detector,
and strip-chart recorder. A data system is recommended for measuring peak areas.
5.6.1 Column 1: 5 Dupont Zorbax-CN, 250 mm long by 4.6 mm ID or equivalent. This
column was used to develop the method performance statements in Section 14.
Alternative columns may be used in accordance with the provisions described in
Section 12.1.
5.6.2 Column 2: 5 i Dupont Zorbax Silica, 250 mm long by 4.6 mm ID or equivalent.
6. REAGENTS
6.1 Reagent water: Reagent water is defined as a water in which an interferent is not observed at the
method detection limit of each parameter of interest.
475
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Methoi! 637
6.2 Methylene chloride, methanol, ethyl ether, and hexane: Distilled-in-glass quality of equivalent.
Ethyl ether must be free of peroxides as indicated by EM Quant test strips (available from
Scientific Products Co., Catalog No. P1126-8 and other suppliers). Procedures recommended for
removal of peroxides are provided with the test strips.
6.3 Sodium sulfate: ACS, granular, anhydrous; heated in a muffle furnace at 40°C overnight.
6.4 Silica gel: Davison Grade 923, 100-120 mesh, dried fur 12 hours at 150°C.
6.5 iN sodium hydroxide: Dissolve 4.0 g of NaOH (ACS) in 100 mL of distilled water.
6.6 iN sulfuric acid: Slowly add 2.8 mL of concentrated H 2 S0 4 (94%) to about 50 mL of distilled
water. Dilute to 100 rnL with distilled water
6.7 Sodium phosphate: Monobasic, ACS grade.
6.8 Sodium phosphate: Dibasic, ACS grade.
6.9 Stock standard solutions (1.00 ig4&L): Stock standard solutions can be prepared from pure•
standard materials or purchased as certified solutions.
6.9.1 Prepare stock standard solutions by accurately weighing about 0.0100 g of pure material.
Dissolve the material in distilled-in-glass quality methylene chloride and dilute to volume
in a IO-mL volumetric flask. Larger volumes can be used at the convenience of the
analyst. If compound purity is certified at 96% or greater, the weight can be used
without correction to calculate the concentration of the stock standard. Commercially
prepared stock standards can be used at any concentration if they are certified by the
manufacturer or by an independent source.
6.9.2 Transfer the stock standard solutions into PTFE-sealed screw-cap bottles. Store at 4°C
and protect from light. Frequently check standard solutions for signs of degradation or
evaporation, especially just prior to preparing calibration standards from them.
6.9.3 Stock standard solutions must be replaced after 6 months, or sooner if comparison with
check standards indicates a problem.
7. C#J.rnRA770N
7.1 Establish liquid chromatographic operating parameters equivalent to those indicated in Table 1.
The liquid chroinatograpbic system can be calibrated using the external standard technique (Section
7.2) or the internal standard technique (Section 7.3).
7.2 External standard calibration procedure:
7.2.1 For each compound of interest, prepare calibration standards at a minimum of three
concentration levels by adding volumes of one or more stock standards to a volumetric
flask and diluting to volume with methylene chloride. One of the external standards
should be at a concentration near, but above, the method detection limit. The other
concentrations should correspond to the range of concentrations expected in the sample
concentrates or should define the working range of the detector.
7.2.2 Using injections of 5 to 20 pL of each calibration standard, tabulate peak height or area
responses against the mass injected. The results can be used to prepare a calibration
curve for each compound. Alternatively, the ratio of the response to the mass injected,
defined as the calibration factor (CF), can be calculated for each compound at each
476
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Method 637
standard concentration. If the relative standard deviation of the calibration factor is less
than 10% over the working range, the average calibration factor can be used in place of
a calibration curve.
7.2.3 The working calibration curve or calibration factor must be verified on each working
shift by the measurement of one or more calibration standards. If the response for any
compound varies from the predicted response by more than ± 10%, the test must be
repeated using a fresh calibration standard. Alternatively, a new calibration curve or
calibration factor must be prepared for that compound.
7.3 Internal standard calibration procedure: To use this approach, the analyst must select one or more
internal standards similar in analytical behavior to the compounds of interest. The analyst must
further demonstrate that the measurement of the internal standard is not affected by method or
matrix interferences. Due to these limitations, no internal standard applicable to all samples can
be suggested.
7.3.1 Prepare calibration standards at a minimum of three concentration levels for each
compound of interest by adding volumes of one or more stock standards to a volumetric
flask. To each calibration standard, add a known constant amount of one or more
internal standards, and dilute to volume with methylene chloride. One of the standards
should be at a concentration near, but above, the method detection limit. The other
concentrations should correspond to the range of concentrations expected in the sample
concentrates or should define the working range of the detector.
7.3.2 Using injections of 5 to 20 ,LL of each calibration standard, tabulate the peak height or
area responses against the concentration for each compound and internal standard.
Calculate response factors (RF) for each compound as follows:
Equation 1
= ( A,)(C , )
where
A, = Response for the parameter to be measured
A = Response for the internal standard
C,, = Concentration of the internal standard, in jigiL
C, = Concentration of the parameter to be measured, in gIL
If the RF value over the working range is constant, less than 10% relative standard
deviation, the RF can be assumed to be invariant and the average RF can be used for
calculations. Alternatively, the results can be used to plot a calibration curve of response
ratios, A 5 IA , against RF.
7.3.3 The working calibration curve or RF must be verified on each working shift by the
measurement of one or more calibration standards. if the response for any compound
varies from the predicted response by more than ± 10%, the test must be repeated using
a fresh calibration standard. Alternatively, a new calibration curve must be prepared for
that compound.
477
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Method 637
7.4 Before using any cleanup procedure, the analyst must process a series of calibration standards
through the procedure to validate elution patterns and the absence of interferences from the
reagents.
8. QUALITY CONTROL
8.1 Each laboratory using this method is required to operate a formal quality control program. The
minimum requirements of this program consist of an initial demonstration of laboratory capability
and the analysis of spiked samples as a continuing check on performance. The laboratory is
required to maintain performance records to define the quality of data that is generated.
8.1 .1 Before performing any analyses, the analyst must demonstrate the ability to generate
acceptable accuracy and precision with this method. This ability is established as
described in Section 8.2.
8 .1.2 In recognition of the rapid advances occurring in chromatography, the analyst is
permitted certain options to improve the separations or lower the cost of measurements.
Each time such modifications to the method are made, the analyst is required to repeat
the procedure in Section 8.2.
8.1.3 The laboratory must spike and analyze a minimum of 10% of all samples to monitor
continuing laboratory performance. This procedure is described in Section 8.4.
8.2 To establish the ability to generate acceptable accuracy and precision, the analyst must perform
the following operations.
8.2.1 Select a representative spike concentration for each compound to be measured. Using
stock standards, prepare a quality control check sample concentrate in methanol, 1000
times more concentrated than the selected concentrations.
8.2.2 Using a pipette, add 1.00 mL of the check sample concentrate to each of a minimum of
four 1000-mL aliquots of reagent water. A representative wastewater may be used in
place of the reagent water, but one or more additional aliquots must be analyzed to
determine background levels, and the spike level must exceed twice the background level
for the test to be valid. Analyze the aliquots according to the method beginning in
Section 10.
8.2.3 Calculate the average percent recovery (R), and the standard deviation of the percent
recovery (s), for the results. Wastewater background corrections must be made before
R and s calculations are performed.
8.2.4 Using the appropriate data from Table 3, determine the recovery and single-operator
precision expected for the method, and compare these results to the values measured in
Section 8.2.3. If the data are not comparable, the analyst must review potential problem
areas and repeat the test.
8.3 The analyst must calculate method performance criteria and define the performance of the
laboratory for each spike concentration and parameter being measured.
8.3.1 Calculate upper and lower control limits for method performance as follows:
Upper Control Limit (tJCL) = R + 3s
Lower Control Limit (LCL) = R - 3s
478
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Method 637
where R and s are calculated as in Section 8.2.3. The UCL and LCL can be used to
construct control charts 6 that are useful in observing trends in performance.
8.3.2 The laboratory must develop and maintain separate accuracy statements of laboratory
performance for wastewater samples. An accuracy statement for the method is defined
as R ± s. The accuracy statement should be developed by the analysis of four aliquots
of wastewater as described in Section 8.2.2, followed by the calculation of R and 5.
Alternatively, the analyst may use four wastewater data points gathered through the
requirement for continuing quality control in Section 8.4. The accuracy statements should
be updated regularly. 6
8.4 The laboratory is required to collect in duplicate a portion of their samples to monitor spike
recoveries. The frequency of spiked sample analysis must be at least 10% of all samples or one
sample per month, whichever is greater. One aliquot of the sample must be spiked and analyzed
as described in Section 8.2. If the recovery for .a particular compound does not fall within the
control limits for method performance, the results reported for that compound in all samples
processed as part of the same set must be qualified as described in Section 13.3. The laboratory
should monitor the frequency of data so qualified to ensure that it remains at or below 5%.
8.5 Before processing any samples, the analyst should demonstrate though the analysis of a 1-L aliquot
of reagent water that all glassware and reagent interferences are under control. Each time a set
of samples is extracted or there is a change in reagents, a laboratory reagent blank should be
processed as a safeguard against laboratory contamination.
8.6 It is recommended that the laboratory adopt additional quality assurance practices for use with this
method. The specific practices that are most productive depend upon the needs of the laboratory
and the nature of the samples. Field duplicates may be analyzed to monitor the precision of the
sampling technique. When doubt exists over the identification of a peak on the chromatogram,
confirmatory techniques such as gas chromatography with a dissimilar column, specific element
detector, or mass spectrometer must be used. Whenever possible, the laboratory should perform
analysis of standard reference materials and participate in relevant performance evaluation studies.
9. SAMPLES COLLECTiON, PRESERVATiON, AND HANDLING
9.1 Grab samples must be collected in glass containers. Conventional sampling practices 7 should be
followed; however, the bottle must not be prerinsed with sample before collection. Composite
samples should be collected in refrigerated glass containers in accordance with the requirements
of the program. Automatic sampling equipment must be as free as possible of plastic and other
potential sources of contamination.
9.2 The samples must be iced or refrigerated at 4°C from the time of collection until extraction.
9.3 Adjust the pH of the sample to 6 to 8 with sodium hydroxide or sulfuric acid immediately after
sampling.
10. SAMPLE EXTRACTiON
10.1 Mark the water meniscus on the side of the sample bottle for later determination of sample
volume. Pour the entire sample into a 2-L separatory funnel. Check the pH of the sample with
479
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Method 637
wide range pH paper and adjust to 6 to 8 with iN sodium hydroxide or iN sulfuric acid.
Dissolve 5 g of monobasic sodium phosphate and 5 g of dibasic sodium phosphate in the sample.
10.2 Add 60 mL of methylene chloride to the sample bottle, seal, and shake 30 seconds to rinse the
inner walls. Transfer the solvent to the separatory funnel and extract the sample by shaking the
funnel for 2 minutes with periodic venting to release excess pressure. Allow the organic layer to
separate from the water phase for a minimum of 10 minutes. If the emulsion interface between
layers is more than one-third the volume of the solvent layer, the analyst must employ mechanical
techniques to complete the phase separation. The optimum technique depends upon the sample,
but may include stirring, filtration of the emulsion through glass wool, centrifugation, or other
physical methods. Collect the methylene chloride extract in a 250-mL Erlenmeyer flask.
10.3 Add a second 60-mL volume of methylene chloride to the sample bottle and repeat the extraction
procedure a second time, combining the extracts in the Erlenmeyer flask. Perform a third
extraction in the same manner.
10.4 Assemble a Kuderna-Danish (K-D) concentrator by attaching a 10-mL concentrator tube to a
500-mL evaporative flask. Other concentration devices or techniques may be used in place of the
K-D if the requirements of Section 8.2 are met.
10.5 Pour the combined extract through a drying column containing about 10 cm of anhydrous sodium
sulfate, and collect the extract in the K-D concentrator. Rinse the Erlenmeyer flask and column
with20 to 30 mL of methylene chloride to complete the quantitative transfer. Once the flask rinse
has passed through the drying column, rinse the column with 30 to 40 mL of methylene chloride.
10.6 Add one or two clean boiling chips to the evaporative flask and attach a three-ball Snyder column.
Prewet the Snyder column by adding about I mL of methylene chloride to the top. Place the K-D
apparatus on a hot water bath, 60 to 65°C, so that the concentrator tube is partially immersed in
the hot water, and the entire lower rounded surface of the flask is bathed with hot vapor. Adjust
the vertical position of the apparatus and the water temperature as required to complete the
concentration in 15 to 20 minutes. At the proper rate of distillation, the balls of the column will
actively chatter but the chambers will not flood with condensed solvent. When the apparent
volume of liquid reaches 1 mL, remove the K-D apparatus and allow it to drain and cool for at
least 10 minutes. If the sample extract requires no cleanup, proceed with Section 10.7. If the
sample extract requires cleanup, proceed to Section 11.
10.7 Remove the macro-Snyder column and rinse the flask and its lower joint into the concentrator tube
with 1 to 2 mL of methylene chloride. Adjust the volume of the extract to 5.0 mL with methylene
chloride. Stopper the concentrator tube and store refrigerated if further processing will not be
performed immediately. If the extract is to be stored longer than 2 days, transfer the extract to
a screw-capped vial with a PTFE-lined cap. If the sample extract requires no further cleanup,
proceed with the liquid chromatographic analysis in Section 12. if the sample requires cleanup,
proceed to Section 11.
10.8 Determine the original sample volume by refilling the sample bottle to the mark and transferring
the water to a 1000-mL graduated cylinder. Record the sample volume to the nearest 5 mL
480
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Method 637
11. CLEANUP AND SEPAR4 T1ON
11.1 Cleanup procedures may not be necessary for a relatively clean sample matrix. The cleanup
procedure recommended in this method has been used for the analysis of various clean waters and
industrial effluents. If particular circumstances demand the use of an alternative cleanup
procedure, the analyst must determine the elution profile and demonstrate that the recovery of
each compound of interest is no less than the recovery values reported in Table 2.
11.2 The following silica gel column cleanup procedure has been demonstrated to be applicable to the
pesticides listed in Table 1.
11.2.1 Add 10 gofsilicagelto 100 mLof ethyl ether and 600iLofreagentwater ina25O-mL
Erlenmeyer flask. Shake vigorously for 15 minutes. Transfer the slurry to a
chromatographic column (silica gel may be retained with a plug of glass wool). Allow
the solvent to elute from the column until the silica gel is almost exposed to the air.
Wash the column with 100 mL of 50% hexane in methylene chloride as before and
discard. Use a column flow of 2 to 2.5 mL/min throughout the wash and elution
profiles.
11.2.2 Quantitatively add the sample extract from Section 10.8 to the head of the column.
Allow the solvent to elute from the column until the silica gel is almost exposed to the
air. Elute the column with 50 mL of 50% hexane in methylene chloride. Discard this
fraction.
11.2.3 Elute the column with 50 mL of methylene chloride (Fraction 1) and collect eluate in a
K-D apparatus. Repeat process with 50 mL of 6% ethyl ether in methylene chloride
(Fraction 2). The TCMTB elutes in Fraction 1 and the MBTS elutes in Fraction 2.
Concentrate each fraction to 5.0 mL as described in Sections 10.6 and 10.7. Proceed
with liquid chromatographic analysis.
11.2.4 The above-mentioned fractions can be combined before concentration at the discretion of
the analyst.
12. LIQUID CHROMATOGRAPHY
12.1 Table 1 summarizes the recommended operating conditions for the liquid chromatograph.
Included in this table are estimated retention times and method detection limits that can be
achieved by this method. Examples of the separations achieved by Column 1 and Column 2 are
shown in Figures 1, 2, and 3. Other columns, chromatographic conditions, or detectors may be
used if the requirements of Section 8.2 are met.
12.2 Calibrate the liquid chromatographic system daily as described in Section 7.
12.3 If the internal standard approach is being used, the analyst must not add the internal standard to
the sample extracts until immediately before injection into the instrument. Mix thoroughly.
12.4 Inject 5 to 20 p.L of the sample extract by completely filling the sample value loop. Record the
resulting peak sizes in areas of peak height units.
12.5 The width of the retention-time window used to make identifications should be based upon
measurements of actual retention-time variations of standards over the course of a day. Three
times the standard deviation of a retention time for a compound can be used to calculate a
481
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Method 637
suggested window size; however, the experience of the analyst should weigh heavily in the
interpretation of chromatograms.
12.6 If the response for the peak exceeds the working range of the system, dilute the extract and
reanalyze.
12.7 if the measurement of the peak response is prevented by the presence of interferences, further
cleanup is required.
13. CALCULATIONS
13.1 Determine the concentration of individual compounds in the sample.
13.1.1 If the external standard calibration procedure is used, calculate the amount of material
injected from the peak response using the calibration curve or calibration factor in Section
7.2.2. The concentration in the sample can be calculated as follows:
Equation 2
( A)(V )
Concentration, pgIL = _____
‘ I, :
where
A = Amowat of material injected, in ng
V 1 = Volwne of extract injected, in pL
V, = Volume of total extract, in 1 iL
V, = Volume of water extracted, in mL
13.1.2 If the internal standard calibration procedure was used, calculate the concentration in the
sample using the response factor (RF) determined in Section 7.3.2 as follows:
Equation 3
Concentration, pgIL = (A 11 )(RF)(V 0 )
where
A, = Response for paraAseter to be measured
4, = Response for the internal standard
I, = Amount of internal standard added to each extract, in pg
V = Volume of water extracted, in L
13.2 Report results in micrograms per liter without correction for recovery data. When duplicate and
spiked samples are analyzed, report all (data obtained with the sample results).
13.3 For samples processed as part of a set where the laboratory spiked sample recovery falls outside
of the control limits in Section 8.3, data for the affected compounds must be labeled as suspect.
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Method 637
14. METHOD PERFORMANCE
14.1 The method detection limit (MDL) is defined as the minimum concentration of a substance that
can be measured and reported with 99% confidence that the value is above zero.’ The MDL
concentrations listed in Table 1 were obtained using reagent water. 1 Similar results were achieved
using representative wastewaters.
14.2 This method has been tested for linearity of recovery from spiked reagent water and has been
demonstrated to be applicable over the concentration range from 10 x MDL to 1000 x MDL.
14.3 In a single laboratory, Battelle Columbus Laboratories, using spiked wastewater samples, the
average recoveries presented in Table 2 were obtained after silica gel cleanup. Seven replicates
of each of two different wastewaters were spiked and analyzed. The standard deviation of the
percent recovery is also included in Table 2.1
483
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Method 637
References
1. “Development of Methods for Pesticides in Wastewaters,” Report for EPA Contract 68-03-2956
(In preparation).
2. ASTM Annual Book of Standards, Part 31, D3694, “Standard Practice for Preparation of Sample
Containers and for Preservation,” American Society for Testing and Materials, Philadelphia, PA,
p. 679, 1980.
3. “Carcinogens - Working with Carcinogens,” Department of Hea1th, Education, and Welfare,
Public Health Service, Center for Disease Control, National Institute for Occupational Safety and
Health, Publication No. 77-206, August, 1977.
4. RØ5} Safety and Health Standards, General Industry,” (29 CFR 1910), Occupational Safety and
Health Administration, OSHA 2206 (Revised, January 1976).
5. “Safety in Academic Chemistry Laboratories,” American Chemical Society Publication,
Committee on Chemical Safety, 3rd Edition, 1979.
6. “Handbook for Analytical Quality Control in Water and Wastewater Laboratories,”
EPA-600/4-79-019, U. S. Environmental Protection Agency, Environmental Monitoring and
Support Laboratory - Cincinnati, Ohio 45268, March 1979.
7. ASTM Annual Book of Standards, Part 31, D3370, “Standard Practice for Sampling Water,”
American Society for Testing and Materials, Philadelphia, PA, p. 76, 1980.
8. Glaser, J. A., et al., “Trace Analysis for Wastewaters,” Environmental Science and Technology,
15, 1426 (1981).
484
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Method 637
Table 1. Chromatographic Conditions and Estimated Detection Limits
Retention Time (Mm.)
MDL
Parameter Column 1 Column 2 (pg/L)
MBTS 6.6 6.3 0.5
TCMTB 9.3 7.9 1.0
Column 1 conditions: Dupont Zorbax-CN, 5 250 by 4.6 mm; 1 mL/min flow; 15/85 methylene
chioride/hexane.
Column 2 conditions: Dupont Zorbax silica, 5 i, 250 by 4.6 mm; 1 mL/min flow; 90/9.5/0.5
hexane/methylene chloride/methanol.
Table 2. Single Laboratory Accuracy and Precision(a)
Sample Background Spike Mean Standard Number of
Parameter Type 1 pg/L pg/L Recovery, % Deviation Replicates
MBTS 1 ND 5 35 23 7
1 ND 100 69 6 7
TCMTB 1 ND 5 69 20 7
1 ND 100 90 2 7
(a) Column 1 conditions were used.
(b) 1 = Municipal sewage effluent
(c) ND = Not detected
485
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Method 637
Figure 1. HPLC-UV Chromatogram of 10 ng Each of MBTS and
TCMTB (Column 1).
MBTS TCMTB
1.5 3.0 4.5 6.0 7.5
9.0
Retention Time (minutes)
10.5
12.0
13.5
15.0
-------
Method 637
MBTS
/
1
I I I I I I I I I I I I I I I I I I
0 1.5 3.0 4.5 6.0 7.5 9.0 10.5 12.0 13.5 15.0
Retenflon Time (minutes)
Figure 2. HPLC-UV Chromatogramof 100 ng of MBTS (Column 2).
487
-------
M9( had 837
TCMTB
I 1 I I
0 1.5 3.0 4.5 6.0 7.5 9.0 10.5 12.0 13.5 15.0
Retention Thne(mh utes)
A .OO2 .36
Agure 3. HPLC-UV Chrornatogram of 100 ng of TCMTB (Column 2).
488
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Method 638
The Determination of Oryzalin
in Municipal and Industrial
Waste waters
-------
Method 638
The Determination of Oryzalin in Municipal and Industrial
Waste waters
1. SCOPE AND APPliCATiON
1.1 This method covers the determination of oryzalin pesticide. The following parameter can be
determined by this method:
Parameters CAS No.
Oryzalin 19044-88-3
1.2 This is a high performance liquid chromatographic (HPLC) method applicable to the determination
of the compound listed above in municipal and industrial discharges as provided under
40 CFR 136.1. Any modification of this method beyond those expressly permitted shall be
considered a major modification subject to application and approval of alternative test procedures
under 40 CFR 136.4 and 136.5.
1.3 The estimated method detection limit (MDL, defined in Section 15) for oryzalin is listed in
Table 1. The MDL for a specific- wastewater may differ from those listed, depending upon the
nature of interferences in the sample matrix.
1.4 This method is restricted to use by or under the supervision of analysts experienced in the use of
liquid chromatography and in the interpretation of liquid chromatograms. Each analyst must
demonstrate the ability to generate acceptable results with this method using the procedure
described in Section 8.2.
1.5 When this method is used to analyze unfamiliar samples for the compound above, compound
identifications should be supported by at least one additional qualitative technique. This method
describes analytical conditions for a second liquid chromatographic column that can be used to
confirm measurements made with the primary column.
2. SUMMARY OF METHOD
2.1 A measured volume of sample, approximately 1 L, is extracted with methylene chloride using a
separatory funnel. The methylene chloride extract is dried and exchanged to acetonitrile during
concentration to a volume of 2 mL or less. Liquid chromatographic conditions are described
which permit the separation and measurement of the compounds in the extract by HPLC-UV. 1
3. INTERFERENCES
3.1 Method interferences may be caused by contaminants in solvents, reagents, glassware, and other
sample-processing hardware that lead to discrete artifacts or elevated baselines in liquid
chromatograms. All of these materials must be routinely demonstrated to be free from
interferences under the conditions of the analysis by running laboratory reagent blanks as
described in Section 8.5.
491
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Method 638
3.1.1 Glassware must be scrupulously cleaned. 2 Clean all glassware as soon as possible after
use by thoroughly rinsing with the last solvent used in it. Follow by washing with hot
water and detergent and thorough rinsing with tap and reagent water. Drain dry, and
heat in an oven or muffle furnace at 400°C for 15 to 30 minutes. Some thermally stable
materials, such as PCBs, may not be eliminated by this treatment. Thorough rinsing with
acetone and pesticide-quality hexane may be substituted for heating. Volumetric wave
should not be heated in a muffle furnace. After drying and cooling, seal and store
glassware in a clean environment to prevent any accumulation of dust or other
contaminants. Store inverted or capped with aluminum foil.
3.1.2 The use of high-purity reagents and solvents helps to minimize interference problems.
Purification of solvents by distillation in all-glass systems may be required.
3.2 Matrix interferences may be caused by contaminants that are coextracted from the sample. The
extent of matrix interferences will vary considerably from source to source, depending upon the
nature and diversity of the industrial complex or municipality being sampled. The cleanup
procedure in Section 11 can be used to overcome these interferences, but unique samples may
require additional cleanup approaches to achieve the MDL listed in Table 1.
4. SAFETY
4.1 The toxicity or carcinogenicity of each reagent used in this method has not been precisely defined;
however, each chemical compound should be treated as a potential health hazard. From this view-
point, exposure to these chemicals must be reduced to the lowest possible level by whatever means
available. The laboratory is responsible for maintaining a current awareness file of OSHA
regulations regarding the safe handling of the chemicals specified in this method. A reference file
of material data handling sheets should also be made available to all personnel involved in the
chemical analysis. Additional references to laboratory safety are available and have been
identifled 3 for the information of the analyst
5. APPARA TUS AND MA TERIALS
5.1 Sampling equipment, for discrete or composite sampling.
5.1.1 Grab-sample bottle: Amber borosiicate or flint glass, 1-L or 1-quart volume, fitted with
screw-caps lined with PTFE. Aluminum foil may be substituted for PTFE if the sample
is not corrosive. If amber bottles are not available, protect samples from light. The
container and cap liner must be washed, rinsed with acetone or methylene chloride, and
dried before use to minimize contamination.
5.1.2 Automatic sampler (optional): Must incorporate glass sample containers for the
collection of a minimum of 250 mL. Sample containers must be kept refrigerated at 4°C
and protected from light during compositing. If the sampler uses a peristaltic pump, a
minimum length of compressible silicone rubber tubing may be used. Before use,
however, the compressible tubing should be thoroughly rinsed with methanol, followed
by repeated rinsings with distilled water to minimize the potential for contamination of
the sample. An integrating flow meter is required to collect flow-proportional
composites.
5.2 Glassware. (All specifications are suggested. Catalog numbers are included for illustration only.)
492
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Method 638
5.2.1 Separatory funnel: 2000-mL, with PTFE stopcock.
5.2.2 Drying column: Chromatographic column 400 mm long by 10 mm ID with coarse fit.
5.2.3 Chromatographic column: 400 mm long by 19 mm ID with 250-mL reservoir at the top
and PTFE stopcock (Kontes K-420290 or equivalent).
5.2.4 Concentrator tube, ICuderna-Danish: 25-mL, graduated (Kontes K-570050-2525 or
equivalent). Calibration must be checked at the volumes employed in the test. A
ground-glass stopper is used to prevent evaporation of extracts.
5.2.5 Evaporative flask, Kuderna-Danish: 250-mL (Kontes K-570001-0250 or equivalent).
Attach to concentrator tube with springs.
5.2.6 Snyder column, Kuderna-Danish: Three-ball macro (Kontes K-503000-0121 or
equivalent).
5.2.7 Snyder column, Kuderna-Danish: Two-ball micro (Kontes K-569001-0219 or•
equivalent).
5.2.8 Vials: Amber glass, 5- to 10-mL capacity with PTFE-lined screw-cap.
5.2.9 Volumetric flask: 2-mL with glass stopper.
5.3 Boiling chips: Approximately 10/41) mesh carborundum. Heat to 400°C for 4 hours or extract
in a Soxhlet extractor with methylene chloride.
5.4 Water bath: Heated, capable of temperature control (±2°C). The bath should be used in a hood.
5.5 Balance: Analytical, capable of accurately weighing to the nearest 0.0001 g.
5.6 Liquid chromatograph: Analytical system complete with liquid chromatograph and all required
accessories including syringes, analytical columns, detector, and strip-chart recorder. A data
system is recommended for measuring peak areas.
5.6.1 Pump: Isocratic pumping system, constant flow.
5.6.2 Column 1: Reversed-phase column, 5 Spherisorb-ODS, 250 mm long by 4.6 mm ID
or equivalent.
5.6.3 Column 2: Reversed-phase column, 5 Lichrosorb RP-2, 250 mm long by 4.6 mm H)
or equivalent.
5.6.4 Detector: Ultraviolet absorbance detector, 254 nm.
6. REAGENTS
6.1 Reagent water: Reagent water is defined as a water in which an interferent is not observed at the
method detection limit of each parameter of interest.
6.2 Methylene chloride, acetone, acetonitrile: Distilled-in-glass quality or equivalent.
6.3 Sodium sulfate: ACS, granular, anhydrous; heated in a muffle furnace at 400°C overnight.
6.4 Sodium hydroxide, IN: Prepared by adding 4 g of sodium hydroxide in distilled water and
diluting to 100 mL.
6.5 Sulfuric acid, iN: Prepared by diluting 2.8 mL of concentrated sulfuric acid to distilled water
and diluting to 100 mL.
493
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Method 638
6.6 Florisil: PR grade (60/100 mesh). Purchase activated at 675°C and store in brown glass bottle.
To prepare for use, place 150 g in a wide-mouth jar and heat overnight at 160 to 170°C. Seal
tightly with FrFE-or aluminum-foil-lined screw-cap and cool to room temperature.
6.7 Stock standard solutions (1.00 ig4iL): Stock standard solutions can be prepared from pure
standard materials or purchased as certified solutions.
6.7.1 Prepare stock standard solutions by accurately weighing about 0.010 g of pure material.
Dissolve the material in distilled-in-glass quality acetonitrile and dilute to volume in a
10-mt volumetric flask. Larger volumes can be used at the convenience of the analyst.
If compound purity is certified at 96% or greater, the weight can be used without
correction to calculate the concentration of the stock standard. Commercially prepared
stock standards can be used at any concentration if they are certified by the manufacturer
or by an independent source.
6.7.2 Transfer the stock standard solutions into PTFE-sealed screw-cap bottles. Store at 4°C
and protect from light. Stock standard solutions should be checked frequently for signs
of degradation or evaporation, especially just prior to preparing calibration standards
from them.
6.7.3 Stock standard solutions must be replaced after 6 months, or sooner if comparison with
check standards indicates a problem.
7. CA BR4 liON
7.1 Establish liquid cbromatographic operating parameters equivalent tO those indicated in Table 1.
The liquid chromatographic system can be calibrated using the external standard technique (Section
7.2) or the internal standard technique (Section 7.3).
7.2 External standard calibration procedure:
7.2.1 For each compound of interest, prepare calibration standards at a minimum of three
concentration levels by adding volumes of one or more stock standards to a volumetric
flask and diluting to volume with acetomtrile. One of the external standards should be
at a concentration near, but above, the method detection limit. The other concentrations
should correspond to the expected range of concentrations found in real samples or
should define the working range of the detector.
7.2.2 Using injections of 2 to 5 pL of each calibration standard, tabulate peak height or area
responses against the mass injected. The results can be used to prepare a calibration
curve for each compound. Alternatively, the ratio of the response to the mass injected,
defined as the calibration factor (CF), can be calculated for each compound at each
standard concentration. If the relative standard deviation of the calibration factor is less
than 10% over the working range, linearity through the origin can be assumed and the
average calibration factor can be used in place of a calibration curve.
7.2.3 The working calibration curve or calibration factor must be verified on each working
shift by the measurement of one or more calibration standards. If the response for any
compound varies from the predicted response by more than ± 10%, the test must be
repeated using a fresh calibration standard. Alternatively, a new calibration curve or
calibration factor must be prepared for that compound.
494
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Method 638
7.3 Internal standard calibration procedure: To use this approach, the analyst must select one or more
internal standards similar in analytical behavior to the compounds of interest. The analyst must
further demonstrate that the measurement of the internal standard is not affected by method or
matrix interferences. Due to these limitations, no internal standard applicable to all samples can
be suggested.
7.3.1 Prepare calibration standards at a minimum of three concentration levels for each
compound of interest by adding volumes of one or more stock standards to a volumetric
flask. To each calibration standard, add a known constant amount of one or more
internal standards, and dilute to volume with acetonitrile. One of the standards should
be at a concentration near, but above, the method detection limit. The other
concentrations should correspond to the expected range of concentrations Ibund in real
samples, or should define the working range of the detector.
7.3.2 Using injections of 2 to 5 L of each calibration standard, tabulate the peak height or
area responses against the concentration for each compound and internal standard.
Calculate response factors (RF) for each compound as follows:
Equation 1
= (A,)(C ,)
(A , ,)(C ,)
where
•
A, =
A,, =
C,, =
C, =
Response for
Response for
Concentration
Concentration
the parameter to be measured
the internal standard
of the internal standard, in ig/L
of the parameter to be measured, in gIL
If the RF value over the working range is constant, less than 10% relative standard
deviation, the RF can be assumed to be invariant and the average RF can be used for
calculations. Alternatively, the results can be used to plot a calibration curve of response
ratios, A,/A 1 against RF.
7.3.3 The working calibration curve or RF must be verified on each working shift by the
measurement of one or more calibration standards. If the response for any compound
varies from the predicted response by more than ± 10%, the test must be repeated using
a fresh calibration standard. Alternatively, a new calibration curve must be prepared for
that compound.
7.4 Before using any cleanup procedure, the analyst must process a series of calibration standards
through the procedure to validate elution patterns and the absence of interferences from the
reagents.
495
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Method 638
8. QuALm CONTROL
8.1 Each laboratory using this method is required to operate a formal quality control program. The
minimum requirements of this program consist of an initial demonstration of laboratory capability
and the analysis of spiked samples as a continuing check on performance. The laboratory is
required to ma’nt in performance records to define the quality of data that is generated.
8.1.1 Before performing any analyses, the analyst must demonstrate the ability to generate
acceptable accuracy and precision with this method. This ability is established as
described in Section 8.2.
8.1.2 In recognition of the rapid advances occurring in chromatography, the analyst is
permitted certain options to improve the separations or lower the cost of measurements.
Each time such modifications to the method are made, the analyst is required to repeat
the procedure in Section 8.2.
8.1.3 The laboratory must spike and analyze a minimum of 10% of all samples to monitor
continuing laboratory performance. This procedure is described in Section 8.4.
8.2 To establish the ability to generate acceptable accuracy and precision, the analyst must perform
the following operations.
8.2.1 Select a representative spike concentration for each compound to be measured. Using
stock standards, prepare a quality control check sample concentrate in methanol, 1000
times more concentrated than the selected concentrations.
8.2.2 Using a pipette, add 1.00 mL of the check sample concentrate to each of a minimum of
four 1000-mL aliquots of reagent water. A representative wastewater may be used in
place of the reagent water, but one or more additional aliquots must be analyzed to
determine background levels, and the spike level must exceed twice the background level
forthetesttobevalid. Analyzethealiquotsaccordingtothemethodbeginningin
Section 10.
8.2.3 Calculate the average percent recovery (R), and the standard deviation of the percent
recovery (s), for the results. Wastewater background corrections must be made before
R and a calculations are performed.
8.2.4 Using the appropriate data from Table 2, determine the recovery and single-operator
precision expected for the method, and compare these results to the values measured in
Section 8.2.3. If the data are not comparable, the analyst must review potential problem
areas and repeat the test.
8.3 The analyst must calculate method performance criteria and define the performance of the
laboratory for each spike concentration and parameter being measured.
8.3.1 Calculate upper and lower control limits for method performance as follows:
Upper Control Limit (UCL) = R + 3s
Lower Control Limit (LCL) = R -3s
where R and a are calculated as in Section 8.2.3. The UCL and LCL can be used to
construct control charts that are useful in observing trends in performance.
496
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Method 638
8.3.2 The laboratory must develop and maintain separate accuracy statements of laboratory
performance for wastewater samples. An accuracy statement for the method is defined
as R ± s. The accuracy statement should be developed by the analysis of four aliquots
of wastewater as described in Section 8.2.2, followed by the calculation of R and s.
Alternatively, the analyst may use four wastewater data points gathered through the
requirement for continuing quality control in Section 8.4. The accuracy statements
should be updated with this method. This ability is established as described regularly. 6
8.4 The laboratory is required to collect in duplicate a portion of their samples to monitor spike
recoveries. The frequency of spiked sample analysis must be at least 10% of all samples or one
sample per month, whichever is greater. One aliquot of the sample must be spiked and analyzed
as described in Section 8.2. If the recovery for a particular compound does not fall within the
control limits for method performance, the results reported for that compound in all samples
processed as part of the same set must be qualified as described in Section 13.3. The laboratory
should monitor the frequency of data so qualified to ensure that it remains at or below 5%.
8.5 Before processing any samples, the analyst should demonstrate through the analysis of a 1-L
aliquot of reagent water that all glassware and reagent interferences are under control. &ch time
a set of samples is extracted or there is a change in reagents, a laboratory reagent blank should
be processed as a safeguard against laboratory contamination.
8.6 It is recommended that the laboratory adopt additional quality assurance practices for use with this
method. The specific practices that are most productive depend upon the needs of the laboratory
and the nature of the samples. Field duplicates may be analyzed to monitor the precision of the
sampling technique. When doubt exists over the identification of a peak on the chromatogram,
confirmatory techniques such as liquid chromatography with a dissimilar column, must be used.
Whenever possible, the laboratory should perform analysis of standard reference materials and
participate in relevant performance evaluation studies.
9. SAMPLES COLLECTiON, PRESERVATiON, AND HANDLING
9.1 Grab samples must be collected in glass containers. Conventional sampling practices 7 should be
followed; however, the bottle must not be prerinsed with sample before collection. Composite
samples should be collected in glass containers in accordance with the requirements of the
program. Automatic sampling equipment must be as free as possible of Tygon and other potential
sources of contamination.
9.2 The samples must be iced or refrigerated at 4°C from the time of collection until extraction.
9.3 Adjust the pH of the sample to 6 to 8 with iN sodium hydroxide or iN sulfuric acid immediately
after sampling.
9.4 All samples must be extracted within 7 days and completely analyzed within 40 days of extraction.
10. SAMPLE EXTRACTiON
10.1 Mark the water meniscus on the side of the sample bottle for later determination of sample
volume. Pour the entire sample into a 2-L separatory funnel. Check the pH of the sample with
wide range pH paper and adjust to 7 with iN sodium hydroxide or iN H 2 S0 4 .
497
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Method 638
10.2 Add 60 mL of methylene chloride to the sample bottle, seal, and shake 30 seconds to rinse the
inner walls. Transfer the solvent to the separatory funnel and extract the sample by shaking the
funnel for 2 minutes with periodic venting to release excess pressure. Allow the organic layer to
separate from the water phase for a minimum of 10 minutes. If the emulsion interface between
layers is inure than one-third the volume of the solvent layer, the analyst must employ mechanical
techniques to complete the phase separation. The optimum technique depends upon the sample,
but may include stirring, filtration of the emulsion through glass wool, centrifugation, or other
physical methods. Collect the methylene chloride extract in a 250-mL Erlenmeyer flask.
10.3 Add a second 60-mL volume of methylene chloride to the sample bottle and repeat the extraction
procedure a second time, collecting the extract. Perform a third extraction in the same manner
and combine the extracts.
10.4 Assemble a Kuderna-Danish (K-D) concentrator by attaching a 25-mL concentrator tube to a
250-inL evaporative flask. Other concentration devices or techniques may be used in place of the
K-I) if the requirements of Section 8.2 are met.
10.5 Pour the combined extract through a drying column containing about 10 cm of anhydrous sodium
sulfate, and collect the extract in the K-D concentrator. Rinse the Erlenmeyer flask and column
with 20 to 30 mL of methylene chloride to complete the quantitative transfer. Once the flask rinse
has passed through the drying column, rinse the column with 30 to 40 mL of methylene chloride.
10.6 Add one or two clean boiling chips to the evaporative flask and attach a three-ball Snyder column.
Prewet the Snyder column by adding about 1 mL methylene chloride to the top. Place the K-D
apparatus on a hot water bath, 60 to 65°C, so that the concentrator tube is partially immersed in
the hot water, and the entire lower rounded surface of the flask is bathed with hot vapor. Adjust
the vertical position of the apparatus and the water temperature as required to complete the
concentration in 15 to 20 minutes. At the proper rate of distillation, the balls of the column will
actively chatter but the chambers will nut flood with condensed solvent. When the apparent
volume of liquid reaches 1 mL, remove the K-D apparatus and allow it to drain and cool for at
least 10 minutes.
10.7 Remove the macro-Snyder column and rinse the flask and its lower joint into the concentrator tube
with 1 to 2 mL of methylene chloride. A 5-mL syringe is recommended for this operation. Add
one or two clean boiling chips and attach a two-ball micro-Snyder column to the concentrator
tube. Prewet the micro-Snyder column with methylene chloride and concentrate the solvent
extract as before. When an apparent volume of 0.5 mL is reached, or the solution stops boiling,
remove the K-D apparatus and allow it to drain and cool for 10 minutes.
10.8 Remove the micro-Snyder column and adjust the volume of the extract to 1.0 mL with methylene
chloride. Stopper the concentrator tube and store refrigerated if further processing will not be
performed immediately. If the extract is to be stored longer than 2o days, transfer the extract to
a screw-capped vial with a Prrt lined cap. If the sample extract requires no further cleanup,
proceed with solvent exchange to acetonitrile as described beginning with Section 11.5. If the
sample requires cleanup, proceed to Section 11.
10.9 Determine the original sample volume by refilling the sample bottle to the mark and transferring
the water to a 1000-mL graduated cylinder Record the sample volume to the nearest 5 mL.
498
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Method 638
11. CLEANUP AND SEPARA nON
11.1 Cleanup procedures may not be necessary for a relatively clean sample matrix. The cleanup
procedure recommended in this method has been used for the analysis of various clean waters and
industrial effluents. If particular circumstances demand the use of additional cleanup, the analyst
must demonstrate that the recovery of each compound of interest is no less than 85%.
11.2 Slurry 10 g of Florisil in 100 mL of methylene chloride which has been saturated with reagent
water. Transfer the slurry to a chromatographic column (Florisil is retained with a plug of glass
wool). Wash the column with 100 mL of methylene chloride. Use a column flow rate of 2 to
2.5 mL/min throughout the wash and elution profiles.
11.3 Add the extract from Section 10.8 to the head of the column. Allow the solvent to elute from the
column until the Florisil is almost exposed to the air. Elute the column with 50 mL of methylene
chloride. Discard this fraction.
11.4 Elute the column with 50 mL of 5% acetone in methylene chloride. Collect this fraction in a K-D
apparatus. Concentrate the column fraction to 1 mL as described in Sections 10.6 and 10.7.
11.5 Add 15 mL of acetonitrile to the concentrate along with one or two clean boiling chips. Attach
a three-ball micro-Snyder column to the concentrator tube. Prewet the micro-Snyder column with
acetonitrile and concentrate the solvent e tract to an apparent volume of 1 mL. Allow the K-D
apparatus to drain and cool for 10 minutes.
11.6 Transfer the liquid to a 2-mL volumetric flask and dilute to the mark with acetonitrile. Mix
thoroughly prior to analysis. If the extracts will not be analyzed immediately, they should be
transferred to PTFE-sealed screw-cap vials and refrigerated. Proceed with the liquid
chromatógraphic analysis.
12. LIQUID CHROMATOGRAPHY
12.1 Table 1 summarizes the recommended operating conditions for the liquid chromatograph.
Included in this table are estimated retention times and method detection limits that can be
achieved by this method. An example of the separations achieved by Column 1 and Column 2
are shown in Figures 1 and 2. Other columns, chromatographic conditions, or detectors may be
used if the requirements of Section 8.2 are met.
12.2 Calibrate the liquid chromatographic system daily as described in Section 7.
12.3 If an internal standard approach is being used, the analyst must not add the internal standard to
sample extracts until immediately before injection into the instrument.
12.4 Inject 2 to 5 iL of the sample extract by completely filling the sample valve loop. Record the
resulting peak sizes in area or peak height units.
12.5 The width of the retention-time window used to make identifications should be based upon
measurements of actual retention-time variations of standards over the course of a day. Three
times the standard deviation of a retention time for a compound can be used to calculate a
suggested window size; however, the experience of the analyst should weigh heavily in the
interpretation of chromatograms.
12.6 If the response for the peak exceeds the working range of the system, dilute the extract and
reanalyze.
499
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Method 638
12.7 If the measurement of the peak response is prevented by the presence of interferences, further
cleanup is required.
13. CALculATioNs
13.1 Determine the concentration of individual compounds in the sample.
13.1.1 If the extenal standard calibration procedure is used, calculate the amount of material
injectedfrom the peak response using the calibration curve or calibration factor in Section
7.2.2. The concentration in the sample can be calculated as follows:
Equation2
Concentration, gIL = (V)(V)
where
A=Amount of material injected, in ng
V 1 = Volume of extract injected, in L
V, = Volwne of total extract, in 1 &L
V = Volume of n ter extracted, in niL
13.1.2
If the internal standard calibration procedure was used, calculate the concentration in the
sample using the response factor (RF) determined in Section 7.3.2 as follows:
Equation 3
Concentration, gIL = (A)(RF)(V)
A, Response for parameter to be measured
4, = Response for the internal standard
I, = Amount of internal standard added to each extract, in ig
= Volume of water extracted, in L
13.2 Report results in micrograms per liter without correction for recovery data. When duplicate and
spiked samples are analyzed, report all data obtained with the sample results.
13.3 For samples processed as part of a set where the laboratory spiked sample recovery falls outside
of the control limits in Section 8.3, data for the affected compounds must be labeled as suspect.
14. METHOD PERFORMANCE
14.1 The method detection limit (MDL) is defined as the minimum concentration of a substance that
can be measured and reported with 99% confidence that the value is above zero. The MDL
concentrations listed in Table 1 were obtained using reagent water. 1 Similar results were achieved
using representative wastewaters.
500
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Method 638
14.2 This method has been tested for linearity of recovery from spiked reagent water and has been
demonstrated to be applicable over the concentration range from 10 x MDL to 1000 X MDL.
14.3 In a single laboratory, Battelle Columbus Laboratories, using spiked wastewater samples, the
average recoveries presented in Table 2 were obtained. Seven replicates of each of two different
wastewaters were spiked and analyzed. The standard deviation of the percent recovery is also
included in Table 2.’
501
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Method 638
References
1 ‘Development of Methods for Pesticides in Wastewaters,’ EPA Contract Report 68-03-2956 (In
preparation).
2. ASTM Annual Book of Standards, Part 31, D3694, ‘Standard Practice for Preparation of Sample
Containers and for Preservation,’ American Society for Testing and Materials, Philadelphia,
Pennsylvania, p. 679, 1980.
3. ‘Carcinogens - Working with Carcinogens,’ Department of Health, Education, and Welfare,
Public Health Service, Center for Disease Control, National Institute for Occupational Safety and
Health, Publication No. 77-206, August, 1977.
4. ‘OSHA Safety and Health Standards, General Industry,’ (29 CFR 1910), Occupational Safety and
Health Adminictration, OSHA 2206 (Revised, January, 1976).
5. ‘Safety in Academic Chemistry Laboratories,’ American Chemical Society Publication,
Committee on Chemical Safety, 3rd Edition, 1979.
6. ‘Handbook for Analytical Quality Control in Water and Wastewater Laboratories,’
EPA-600/4-79-019, U. S. Environmental Protection Agency, Environmental Monitoring and
Support Laboratory - Cincinnati, Ohio 45268, March, 1979.
7. ASTM Anmial Book of Standards, Part 31, D3370, ‘Standard Practice for Sampling Water,’
American Society for Testing and Materials, Philadelphia, Pennsylvania, P. 76, 1980.
8. Glaser, J. A. et al, ‘Trace Analysis for Wastewaters,’ Environmental Science and Technology,
15, 1426 (1981).
502
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Method 638
Table 1. Chromatographic Conditions and Method Detection Limits
Retention Time (Mm..) MDL
Parameter Column 1 Column 2 (pg/L)
Oryzalin 6.2 10.7 0.5
Column I conditions: Spherisorb-ODS, 5 i, 250 mm long by 4.6 mm ID; 1 mL/min flow; 40/60
acetonitrile/water. A UV detector was used with this column to determine the MDL.
Column 2 conditions: Lichrosorb RP-2, 5 , 250 mm long by 4.6 mm ID; 1 mL/min flow;
50/50 acetonitrilelwater.
Table 2. Single-Laboratory Accuracy and Precision (a)
Sample Spike Mean Standard Number
Type (b) Background Level Recovery (%) Deviation (%) of Replicates
Parameter pg/L pg/L
Oryzalin 1 4 10 106 6 7
2 40 200 100 10 7
(a) Column 1 conditions were used.
(b) 1 = Relevant industrial wastewater diluted 1000:1 with municipal sewage effluent
2 = Relevant industrial wastewater diluted 100:1 with municipal sewage effluent
503
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Method 638
0 2.0 4.0 6.0 8.0 10.0 12.0 14.0 16.0
R entIan Tkne (minutes)
s2-o .a
Figure 1. HPLC-UV Chromatogram of 10 ng of Oryzalin (Columni).
504
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Method 638
O yza in
/
I ’I I I I I I I I
0 2.0 4.0 6.0 8.0 10.0 12.0 14.0 16.0
Retention Time (minutes)
Figure 2. HPLC-UV Chromatogram of 250 ng of Oryzalin (Column 2).
505
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Method 639
The Determination of
Bendiocarb in Municipal and
Industrial Waste waters
-------
Method 639
The Determination of Bendiocarb in Municipal and Industrial
Waste waters
1. SCOPE AND APPLiCATION
1.1 This method covers the determination of bendiocarb pesticide. The following parameter can be
determined by this method:
Parameter CAS No.
Bendiocarb 22781-23-3
1.2 This is a high performance liquid chromatographic (HPLC) method applicable to the determination
of the compound listed above in municipal and industrial discharges as provided under
40 CFR 136.1. When this method is used to analyze unfamiliar samples for the compound above,
compound identifications should be supported by at least one additional qualitative technique.
This method describes analytical conditions for a second liquid chromatographic column that can
be used to confirm measurements made with the primary column.
1.3 The method detection limit (MDL, defined in Section 15) for bendiocarb is listed in Table 1. The
MDL for a specific wastewater may differ from those listed, depending upon the nature of
interferences in the sample matrix.
1.4 Any modification of this method beyond those expressly permitted shall be considered a major
modification subject to application and approval of alternative test procedures under 40 CFR 136.4
and 136.5.
1.5 This method is restricted to use by or under the supervision of analysts experienced in the use of
liquid chromatography and in the interpretation of liquid chromatograms. Each analyst must
demonstrate the ability to generate acceptable results with this method using the procedure
described in Section 8.2.
2. SUMMARY OF METHOD
2.1 A measured volume of sample, approximately 1 L, is extracted with methylene chloride using a
separatory funnel. The methylene chloride extract is dried and exchanged to acetonitrile during
concentration to a volume of 2 mL or less. Liquid chromatographic conditions are described
which permit the separation and measurement of the compounds in the extract by HPLC-UV. 1
3. INTERFERENCES
3.1 Method interferences may be caused by contaminants in solvents, reagents, glassware, and other
sample-processing hardware that lead to discrete artifacts or elevated baselines in liquid
chromatograms. All of these materials must be routinely demonstrated to be free from
interferences under the conditions of the analysis by running laboratory reagent blanks as
described in Section 8.5.
509
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Method 639
3.1.1 Glassware must be scrupulously cleaned. 2 Clean all glassware as soon as possible after
use by rinsing with the last solvent used in it. This should be followed by detergent
washing with hot water and rinses with tap and distilled water. It should then be drained
dry, and heated in a muffle furnace at 400°C for 15 to 30 minutes. Some thermally
stable materials, such as PCBs, may not be eliminated by this treatment. Solvent rinses
with acetone and pesticide-quality hexane may be substituted for the heating. After
drying and cooling, glassware should be sealed and stored in a clean environment to
prevent any accumulation of dust or other contaminants. Store inverted or capped with
aluminum foil.
3.1.2 The use of high-purity reagents and solvents helps to minimize interference problems.
Purification of solvents by distillation in all-glass systems may be required.
3.2 Matrix interferences may be caused by contaminants that are coextracted from the sample. The
extent of matrix interferences will vary considetably from source to source, depending upon the
n nre aed diversity of the industrial complex or municipality being sampled. Unique samples
may require additional cleanup approaches to achieve the MDL listed in Table 1.
4. SAFETY
4.1 The toxicity or carcinogenicity of each reagent used in this method has not been precisely defined;
however, each chemical compound should be treated as a potential health hazard. From this
viewpoint, exposure to these chemicals must be reduced to the lowest possible level by whatever
means available. The laboratory is responsible for maintaining a current awareness file of OSHA
regulations regarding the safe handling of the chemicals specified in this method. A reference file
of material data handling sheets should also be made available to all personnel involved in the
chemical analysis. Additional references to laboratory safety are available and have been
identified ’ 4 for the information of the analyst.
5. Am’Ai TUS AND MA TERIALS
5.1 Sampling equipment, for discrete or composite sampling.
5.1.1 Grab-sample bottle: Amber glass, 1-L or 1-quart volume, fitted with screw-caps lined
with l’TFE. Foil may be substituted for PTFE if the sample is not corrosive. If amber
bottles are not available, protect samples from light. The container and cap liner must
be washed, rinsed with acetone or methylene chloride, and dried before use to minimize
coi* rnin*ion.
5.1.2 Automatic sampler (optional): Must incorporate glass sample containers for the
collection of a minimum of 250 mL. Sample containers must be kept refrigerated at 4°C
and protected from light during compositing. If the sampler uses a peristaltic pump, a
minimum length of compressible silicone rubber tubing may be used. Before use,
however, the compressible tubing should be thoroughly rinsed with methanol, followed
by repeated nnsings with distilled water to minimize the potential for contamination of
the sample. An integrating flow meter is required to collect flow-proportional
composites.
510
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Method 639
5.2 Glassware. (All specifications are suggested. Catalog numbers are included for illustration only.)
5.2.1 Separatory funnel: 2000-mL, with F1’FE stopcock.
5.2.2 Drying column: Chromatographic column 400 mm long by 10 mm ID with coarse fit.
5.2.3 Chromatographic column: 400 mm long by 19 mm ID with 250-mL reservoir at the top
and PTFE stopcock (Kontes K-420290 or equivalent).
5.2.4 Concentrator tube, Kuderna-Danish: 10-mL, graduated (Kontes K-570050-1025 or
equivalent). Calibration must be checked at the volumes employed in the test. A
ground-glass stopper is used to prevent evaporation of extracts.
5.2.5 Evaporative flask, Kuderna-Danish: 500-mL (Kontes K-570001-0500 or equivalent).
Attach to concentrator tube with springs.
5.2.6 Snyder column, Kuderna-Danish: Three-ball macro (Kontes K-503000-0121 or
equivalent).
5.2.7 Snyder column, Kuderna-Danish: Two-ball micro (Kontes K-569001-0219 or
equivalent).
5.2.8 Vials: Amber glass, 5- to 10-mL capacity with PTFE-lined screw-cap.
5.3 Boiling chips: Approximately 10/40 mesh carborundum. Heat to 400°C for 4 hours or extract
in a Soxhlet extractor with methylene chloride.
5.4 Water bath: Heated, capable of temperature control (±2°C). The bath should be used in a hood.
5.5 Balance: Analytical, capable of accurately weighing to the nearest 0.0001 g.
5.6 Liquid chromatograph: Analytical system complete with liquid chromatograph and all required
accessories including syringes, analytical columns, detector, and strip-chart recorder. A data
system is recommended for measuring peak areas.
5.6.1 Pump: Isocratic pumping system, constant flow.
5.6.2 Column 1: Reversed-phase column, 5 Spherisorb-ODS, 250 mm long by 4.6mm ID
or equivalent.
5.6.3 Column 2: Reversed-phase column, 5 Lichrosorb RP-2, 250 mm long by 4.6 mm ID
or equivalent.
5.6.4 Detector: Ultraviolet absorbance detector, 254 nm.
6. REAGENTS
6.1 Reagent water: Reagent water is defined as a water in which an interferent is not observed at the
method detection limit of each parameter of interest.
6.2 Methylene chloride, methanol acetonitrile: Distilled-rn-glass quality or equivalent.
6.3 Sodium sulfate: ACS, granular, anhydrous; heated in a muffle furnace at 400°C overnight.
6.4 Sodium hydroxide, iN: Prepare by adding 4 g of sodium hydroxide to distilled water and diluting
to iOOmL.
6.5 Sulfuric acid, iN: Prepare by adding 2.8 mL of concentrated sulfuric acid to distilled water and
diluting to lOOmL.
511
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Method 639
6.6 Florisil: PR grade (60/100 mesh). Purchase activated at 675°C and store in a brown glass bottle.
To prepare for use, place 150 g in a wide-mouth jar and heat overnight at 160 to 170°C. Seal
tightly with PTFE- or aluminum-foil-lined cap and cool to room temperature.
6.7 Stock standard solutions (1.00 ig/ L): Stock standard solutions can be prepared from pure
standard materials or purchased as certified solutions.
6.7.1 Prepare stock standard solutions by accurately weighing about 0.0100 g of pure material.
Dissolve the material in distilled-in-glass quality methanol and dilute to volume in a 10-
mL volumetric flask. Larger volumes can be used at the convenience of the analyst. If
compound purity is certified at 96% or greater, the weight can be used without correction
to calculate the concentration of the stock standard. Commercially prepared stock
standards can be used at any concentration if they are certified by the manufacturer or
by an independent source.
6.7.2 Transfer the stock standard solution into PTFE-sealed screw-cap bottles. Store at 4°C
and protect from light. Stock standard solution should be checked frequently for signs
of degradation or evaporation, especially just prior to preparing calibration standards
from them.
6.7.3 Stock standard solution must be replaced after 6 months, or sooner if comparison with
quality control check standards indicates a problem.
7. CALIBRA TION
7.1 Establish liquid chromatographic operating parameters equivalent to those indicated in Table 1.
The liquid chromatographic system can be calibrated using the external standard technique (Section
7.2) or the internal standard technique (Section 7.3).
7.2 External standard calibration procedure:
7.2.1 For the compound of interest prepare calibration standards at a minimum of three
concentration levels by adding volumes of one or more stock standards to a volumetric
flask and diluting to volume with acetonitrile. One of the external standards should be
at a concentration near, but above, the method detection limit. The other concentrations
should correspond to the expected range of concentrations found in real samples or
should define the working range of the detector.
7.2.2 Using injections of 2 to 5 L of each calibration standard, tabulate peak height or area
responses against the mass injected. The results can be used to prepare a calibration
curve for bendiocarb. Alternatively, the ratio of the response to the mass injected,
defined as the calibration factor (CF), can be calculated at each standard concentration.
If the relative standard deviation of the calibration factor is less than 10% over the
working range, linearity through the origin can be assumed and the average calibration
factor can be used in place of a calibration curve.
7.2.3 The working calibration curve or calibration factor must be verified on each working day
by the measurement of one or more calibration standards. If the response for bendiocarb
varies from the predicted response by more than ±10%, the test must be repeated using
a fresh calibration standard. Alternatively, a new calibration curve or calibration factor
must be prepared.
512
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Method 639
7.3 Internal standard calibration procedure: To use this approach, the analyst must select one or more
internal standards similar in analytical behavior to bendiocarb. The analyst must further
demonstrate that the measurement of the internal standard is not affected by method or matrix
interferences. Due to these limitations, no internal standard applicable to all samples can be
suggested.
7.3.1 Prepare calibration standards at a minimum of three concentration levels for bendiocarb
by adding volumes of one or more stock standards to a volumetric flask. To each
calibration standard, add a known constant amount of one or more internal standards, and
dilute to volume with acetonitrile. One of the standards should be at a concentration
near, but above, the method detection limit. The other concentrations should correspond
to the expected range of concentrations found in real samples, or should define the
working range of the detector.
7.3.2 Using injections of 2 to 5 L of each calibration standard, tabulate the peak height or
area responses against the concentration for bendiocarb and internal standard. Calculate
response factors (RE) as follows:
Equation 1
RF ( A,)(C )
(4,)(C ,)
where
A, = Response for the parameter to be measured
AL, = Response for the internal standard
C 1 , = Concentration of the internal standard, in gIL
C, = Concentration of the parameter to be measured, in p g/L
If the RE value over the working range is constant, less than 10% relative standard
deviation, the RE can be assumed to be invariant and the average RE can be used for
calculations. Alternatively, the results can be used to plot a calibration curve of response
ratios, A ,/A 11 , against RE.
7.3.3 The working calibration curve or RE must be verified on each working day by the
measurement of one or more calibration standards. If the response for bendiocarb varies
from the predicted response by more than ±10%, the test must be repeated using a fresh
calibration standard. Alternatively, a new calibration curve must be prepared.
7.4 Before using any cleanup procedure, the analyst must process a series of calibration standards
through the procedure to validate elution patterns and the absence of interferences from the
reagents.
8. QuAurY CONTROL
8.1 Each laboratory using this method is required to operate a formal quality control program. The
minimum requirements of this program consist of an initial demonstration of laboratory capability
and the analysis of spiked samples as a continuing check on performance. The laboratory is
required to maintain performance records to define the quality of data that is generated.
513
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Method 639
8.1.1 Before performing any analyses, the analyst must demonstrate the ability to generate
acceptable accuracy and precision with this method. This ability is established as
described in Section 8.2.
8.1.2 In recognition of the rapid advances occurring in chromatography, the analyst is
permitted certain options to improve the separations or lower the cost of measurements.
Each time such modifications to the method are made, the analyst is required to repeat
the procedure in Section 8.2.
8.1.3 The laboratory must spike and analyze a minimum of 10% of all samples to monitor
continuing laboratory performance. This procedure is described in Section 8.4.
8.2 To establish the ability to generate acceptable accuracy and precision, the analyst must perform
the following operations.
8.2.1 Select a representative spike concentration for each compound to be measured. Using
stock standirds, prepare a quality control check sample concentrate in methanol 1000
times more concentrated than the selected concentrations.
8.2.2 Using a pipette, add 1.00 mL of the check sample concentrate to each of a minimum of
four 1000-inL aliquots of reagent water. A representative wastewater may be used in
place of the reagent water, but one or more additional aliquots must be analyzed to
determine background levels, and the spike level must exceed twice the background level
for the test to be valid. Analyze the aliquots according to the method beginning in
Section 10.
8.2.3 Calculate the average percent recovery (R), and the standard deviation of the percent
recovery (s), for the results. Wastewater background corrections must be made before
R and s calculations are performed.
8.2.4 Using the propriate data from Table 2, determine the recovery and single-operator
precision expected for the method, and compare these results to the values measured in
Section 8.2.3. If the data are not comparable, the analyst must review potential problem
areas and repeat the test.
8.3 The analyst must calculate method performance criteria and define the performance of the
laboratory fix each spike concentration and parameter being measured.
8.3.1 Calculate upper and lower control lmits for method performance as follows:
Upper Control Limit (UCL) = R +3s
Lower Control Limit (LCL) = R -3s
Where R and a are calculated as in Section 8.2.3. The UCL and LCL can be used to
construct control charts’ that are useful in observing trends in performance.
8.3.2 The laboratory must develop and maintain separate accuracy statements of laboratory
performance for wastewater samples. An accuracy statement for the method is defined
as R ± s. The accuracy statement should be developed by the analysis of four aliquots
of wastewater as described in Section 8.2.2, followed by the calculation of R and s.
Alternatively, the analyst may use four wastewater data points gathered through the
514
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Method 639
requirement for continuing quality control in Section 8.4. The accuracy statements
should be updated with this method. This ability is established as described regularly. 6
8.4 The laboratory is required to collect in duplicate a portion of their samples to monitor spike
recoveries. The frequency of spiked sample analysis must be at least 10% of all samples or one
sample per month, whichever is greater. One aliquot of the sample must be spiked and analyzed
as described in Section 8.2. If the recovery for a particular compound does not fall within the
control limits for method performance, the results reported for that compound in all samples
processed as part of the same set must be qualified as described in Section 13.3. The laboratory
should monitor the frequency of data so qualified to ensure that it remains at or below 5%.
8.5 Before processing any samples, the analyst should demonstrate through the analysis of a 1-L
aliquot of reagent water that all glassware and reagent interferences are under control. Each time
a set of samples is extracted or there is a change in reagents, a laboratory reagent blank should
be processed as a safeguard against laboratory contamination.
8.6 It is recommended that the laboratory adopt additional quality assurance practices for use with this
method. The specific practices that are most productive depend upon the needs of the laboratory
and the nature of the samples. Field duplicates may be analyzed to monitor the precision of the
sampling technique. When doubt exists over the identification of a peak on the chromatogram,
confirmatory techniques such as liquid chromatography with a dissimilar column must be used.
Whenever possible, the laboratory should perform analysis of standard reference materials and
participate in relevant performance evaluation studies.
9. SAMPLES COLLECTiON, PRESERVATiON, AND HANDLING
9.1 Grab samples must be collected in glass containers. Conventional sampling practices 7 should be
followed; however, the bottle must not be prerinsed with sample before collection. Composite
samples should be collected in refrigerated glass containers in accordance with the requirements
of the program. Automatic sampling equipment must be as free as possible of Tygon and other
potential sources of contamination.
9.2 The samples must be iced or refrigerated at 4°C from the time of collection until extraction.
9.3 Adjust the pH of the sample to 6 to 8 with iN sodium hydroxide or iN sulfuric acid immediately
after sampling.
10. SAMPLE EXTRACTION
10.1 Mark the water meniscus on the side of the sample bottle for later determination of sample
volume. Pour the entire sample into a 2-L separatory funnel. Check the pH of the sample with
wide range pH paper and adjust to 7 with iN sodium hydroxide or iN H 2 S0 4 .
10.2 Add 60 mL of methylene chloride to the sample bottle, seal, and shake 30 seconds to rinse the
inner walls. Transfer the solvent to the separatory funnel and extract the sample by shaking the
funnel for 2 minutes with periodic venting to release excess pressure. Allow the organic layer to
separate from the water phase for a minimum of 10 minutes. If the emulsion interface between
layers is more than one-third the volume of the solvent layer, the analyst must employ mechanical
techniques to complete the phase separation. The optimum technique depends upon the sample,
515
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Method 639
but may include stirring, filtration of the emulsion through glass wool, centrifugation, or other
physical methods. Collect the methylene chloride extract in a 250-mL Erlenmeyer flask.
10.3 Add a second 60-mL volume of methylene chloride to the sample bottle and repeat the extraction
procedure a second time, collecting the extract. Perform a third extraction in the same m inner
and collect the extract
10.4 Assemble a Kuderna-D2nith (K-D) concentrator by attaching a lO-mL concentrator tube to a 250-
mL evaporative flask. Other concentration devices or techniques may be used in place of the K-D
if the requirements of Section 8.2 are met.
10.5 Pour the ombined extract through a drying column containing about 10 cm of anhydrous sodium
sulfate, and collect the extract in the K-D concentrator. Rinse the Erlenmeyer flask and column
with 20 to 30 mL of methylene chloride to complete the quantitative transfer. Once the flask rinse
has passed through the drying column, rinse the column with 30 to 40 mL of methylene chloride.
10.6 Add one or two clean boiling chips to the evaporative flask and attach a macro-Snyder column.
Prewct the Snyder column by adding about 1 mL methylene chloride to the top. Place the K-D
apparatus on a hot water bath, 60 to 65°C, so that the concentrator tube is partially immersed in
the hot water , and the entire lower rounded surface of the flask is bathed with hot vapor. Adjust
the vertical position of the apparatus and the water temperature as required to complete the
concentrationin l5to20minutes. Attheproper rateofdistillation,theballsofthecolumnwil
actively chatter but the chambers will not flood with condensed solvent. When the apparent
volume of liquid reaches 1 mL, remove the K-D apparatus and allow it to drain and cool for at
least 10 minutes.
10.7 Remove the macro-Snyder column and rinse the flask and its lower joint into the concentrator tube
with 1 to 2 mL of methylene chloride. A 5-mL syringe is recommended for this operation. Add
one or two clean boiling chips and 2itath a two-ball micro-Snyder column to the concentrator
tube. Prcwet the micro-Snyder column with methylene chloride and concentrate the solvent
extract as bdbre. When an apparent volume.of 0.5 mL is reached, or the solution stops boiling,
remove the K-D apparatus and allow it to drain and cool for 10 minutes.
10.8 Remove the micro-Snyder column and adjust the volume of the extract to 1.0 mL with methylene
chloride. Stopper the concentrator tube and store refrigerated if further processing will not be
performed immediately. Iftheextractistobestoredlongerthan2days,transfertheextractto
a screw-capped vial with a PTFE-lined cap. If the sample extract requires no further cleanup,
proceed with solvent exchange to acetonitrile as described beginning in Section 11.5. If the
sample requires cleanup, proceed to Section 11.
10.9 Determine the original sample volume by refilling the sample bottle to the mark and transferring
the water to a 1000-mL graduated cylinder. Record the sample volume to the nearest 5 mL.
11. CLE4N& AND SEPARA 770N
11.1 Cleanup procedures may not be necessary for a relatively clean sample matrix. The cleanup
procedure recommended in this method has been used for the analysis of various clean waters and
industrial effluents. If particular circumstances demand the use of additional cleanup, the analyst
must demonstrate that the recovery of each compound of interest is no less than 65%.
11.2 Slurry 10 g of Florisil in 100 mL of methylene chloride which has been saturated with reagent
water. Transfer the slurry to a chromatographic column. Wash the column with 100 mL of
516
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Method 639
methylene chloride. Use a column flow rate of 2 to 2.5 mL/min throughout the wash and elution
profiles.
11.3 Add the extract from Section 10.8 to the head of the column. Allow the solvent to elute from the
column until the Florisil is almost exposed to the air. Elute the column with 50 mL of methylene
chloride. Discard this fraction.
11.4 Elute the column with 50 mL of 5% acetone in methylene chloride. Collect this fraction in a K-D
apparatus. Concentrate the column fraction to 1 mL as described in Sections 10.6 and 10.7.
11.5 Add 10 mL of acetonitrile to the concentrate along with one or two clean boiling chips. Attach
a three-ball micro-Snyder column to the concentrator tube. Prewet the micro-Snyder column with
acetonitrile and concentrate the solvent extract to an apparent volume of 1 mL. Allow the K-D
apparatus to drain and cool for 10 minutes.
11.6 Transfer the liquid to a 2-mL volumetric flask and dilute to the mark with acetonitrile. Mix
thoroughly prior to analysis. If the extracts will not be analyzed immediately, they should be
transferred to PTFE-sealed screw-cap vials and refrigerated. Proceed with the liquid
chromatographic analysis.
12. LIQuID CHROMATOGRAPHY
12.1 Table 1 summarizes the recommended operating conditions for the liquid chromatograph.
Included in this table are estimated retention times and method detection limits that can be
achieved by this method. An example of the separations achieved by Column 1 and Column 2
are shown in Figures 1 and 2. Other columns, chromatographic conditions, or detectors may be
used if the requirements of Section 8.2 are met.
12.2 Calibrate the liquid chromatographic system daily as described in Section 7.
12.3 If an internal standard approach is being used, the analyst must not add the internal standard to
sample extracts until immediately before injection into the instrument. Mix thoroughly.
12.4 Inject 2 to 5 iiL of the sample extract by completely filling the sample valve loop. Record the
resulting peak sizes in area or peak height units.
12.5 The width of the retention-time window used to make identifications should be based upon
measurements of actual retention-time variations of standards over the course of a day. Three
times the standard deviation of the retention time for a compound can be used to calculate a
suggested window size; however, the experience of the analyst should weigh heavily in the
interpretation of chromatograms.
12.6 If the response for the peak exceeds the working range of the system, dilute the extract and
reanalyze.
12.7 If the measurement of the peak response is prevented by the presence of interferences, further
cleanup is required.
13. CALCULATIONS
13.1 Determine the concentration of individual compounds in the sample.
517
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Method 639
13.1.1 If the external standard calibration procedure is used, calculate the amount of material
injected from the peak response using the calibration curve or calibration factor in
Section 7.2.2. The concentration in the sample can be calculated as follows:
Equation 2
Concentration, ig/L = _____
where
A = Amount of material injected, in ng
= Voiwne of extract injected, in ,vL
V 1 = Voiwne of total extract, in zL
V = Volume of water extracted, in mL
13.1.2 If the internal standard calibration procedure was used, calculate the concentration in the
sample using the response factor (RF) determined in Section 7.3.2 as follows:
- Equation 3
Concentration, tg/L = (A 1 ,)(RF)(V 0 )
•
where
A, =
A =
=
V =
Response for parameter to be measured
Response for the internal standard
Amount of internal standard added to each extract,
Voiwne of water extracted, inL
in
ig
13.2 Report results in micrograms per liter without correction for recovery data. When duplicate and
spiked samples are analyzed, report all data obtained with the sample results.
13.3 For samples processed as part of a set where the laboratory spiked sample recovery falls outside
of the control limits in Section 8.3, data for the affected compounds must be labeled as suspect.
14. METHOD PERFORMANCE
14.1 The method detection limit (MDL) is defined as the minimum concentration of a substance that
can be measured and reported with 99% confidence that the value is above zero. 8 The MDL
concentrations listed in Table 1 were obtained using reagent water. 1 Similar results were achieved
using representative wastewaters.
14.2 This method has been tested for linearity of recovery from spiked reagent water and has been
demonstrated to be applicable over the concentration range from 10 X MDL to 1000 X MDL.
518
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Method 639
14.3 In a single laboratory, Battelle Columbus Laboratories, using spiked wastewater samples, the
average recoveries presented in Table 2 were obtained. Seven replicates of each of two different
wastewaters were spiked and analyzed. The standard deviation of the percent recovery is also
included in Table 2.1
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Method 639
References
1. “Development of Methods for Pesticides in Wastewaters,” EPA Contract Report 68-03-2956 (In
preparation).
2. ASTM Annual Book of Standards, Part 31, D3694, “Standard Practice for Preparation of Sample
Containers and for Preservation,” American Society for Testing and Materials, Philadelphia,
Pennsylvania, p. 679, 1980.
3. Carcinogens - Working with Carcinogens,” Department of Health, Education, and Welfare, Public
Health Service, Center for Disease Control, National Institute for Occupational Safety and Health,
Publication No. 77-206, August, 1977.
4. “OSHA Safety and Health Standards, General Industry,” (29 CFR 1910), Occupational Safety and
Health Administration, OSHA 2206 (Revised, January, 1976).
5. “Safety in Academic Chemistry Laboratories,” American Chemical Society Publication,
Committee on Chemical Safety, 3rd Edition, 1979.
6. “Handbook for Analytical Quality Control in Water and Wastewater Laboratories,” EPA-600/4-
79-019, U.S. Environmental Protection Agency, Environmental Monitoring and Support
Laboratory - Cincinnati, Ohio, March, 1979.
7. ASTM Annual Book of Standards, Part 31, D3370, “Standard Practice for Sampling Water,”
American Society for Testing and Materials, Philadelphia, Pennsylvania, p. 76, 1980.
8. Glaser, J. A. et al, “Trace Analysis for Wastewaters,” Environmental Science and Technology,
15, 1426, (1981).
520
-------
Method 639
Table 1. Chromatographic Conditions and Method Detection Limits
Parameter Retention Time(Minutes) Method Detection Limits
Column 1 Column 2 (pg/L)
Bendiocarb 9.3 6.0 1.8
Column 1 conditions: Spherisorb-ODS, 5 , 250 mm long by 4.6 mm ID; 1 mL/min flow; 40/60
acentonitrile/water.
Column 2 conditions: Lichrosorb RP-2, 5 t, 250 mm long by 4.6 mm ID; 1 mL/min flow; 50/50
acetonitrilelwater.
Table 2. Single-Laboratory Accuracy and Precision (a)
Parameter Average Percent Relative Standard Spike Level No. of Matrix
Recovery Deviation (%) (pgJL) Analyses Type (b)
Bendiocarb 65 35.6 8 7 1
70 5.7 80 7 1
(a) Column 1 conditions were used.
(b) I = Relevant industrial wastewater
521
-------
?.bffiod 639
Figure 1. HPLC-UV Chromatogram of 10 ng of Bendiocarb (Column 1).
Bendiocarb
0 2.0 4.0 6.0 8.0 10.0 12.0 14.0
Retention Time (minutes)
16.0
522
-------
Method 639
— Bendiocarb
I I I I I I I I I I
0 2.0 4.0 6.0 8.0 10.0 12.0 14.0
Retention Time (minutes)
A52-002-3
Figure 2. HPLC-UV Chromatograrn of 600 ng of Bendiocarb (Column 2).
523
-------
Method 640
The Determination of
Mercap tobenzo thiazole in
Municipal and Industrial
Waste waters
-------
526
-------
Method 640
The Determination of Mercaptobenzothiazole in Municipal and
Industrial Waste waters
1. SCOPE AND APPliCATION
1.1 This method covers the determination of mercaptobenzothiazole. The following parameter can
be determined by this method:
Parameter CAS No.
Mercaptobenzothiazole 149-30-4
1.2 This is a high performance liquid chromatographic (HPLC) method applicable to the determination
of the compound listed above in municipal and industrial discharges as provided under
40 CFR 136.1. Any modification of this method beyond those expressly permitted shall be
considered a major modification subject to application and approval of alternative test procedures
under4OCFR 136.4 and 136.5.
1.3 The method detection limit (MDL, defined iii Section 14) for each parameter is listed in Table 1.
The MDL for a specific wastewater may differ from those listed, depending upon the nature of
interferences in the sample matrix.
1.4 The sample extraction and concentration steps in this method are essentially the same as in certain
other 600-series methods. Thus, a single sample may be extracted to measure the compounds
included in the scope of the methods. When cleanup is required, the concentration levels must
be high enough to permit selecting aliquots, as necessary, in order to apply appropriate cleanup
procedures.
1.5 This method is restricted to use by or under the supervision of analysts experienced in the use of
liquid chromatography and in the interpretation of liquid chromatograms. Each analyst must
demonstrate the ability to generate acceptable results with this method using the procedure
described in Section 8.2.
1.6 When this method is used to analyze unfamiliar samples for any or all of the compounds above,
compound identifications should be supported by at least one additional qualitative technique. This
method describes analytical conditions for a second liquid chromatographic column that can be
used to confirm measurements made with the primary column.
2. SUMMARY OF METHOD
2.1 A measured volume of sample, approximately 1 L, is extracted with methylene chloride using a
separatory funnel. The methylene chloride extract is dried and concentrated to 1.0 rnL.
Liquid chromatographic conditions are described which permit the separation and measurement
of the compounds in the extract by high performance liquid chromatography with ultraviolet
detection.
2.2 This method provides a silica gel column cleanup procedure to aid in the elimination of
interferences which may be encountered.
527
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Method 640
3. INTERFERENCES
3.1 Method interferences may be caused by contaminants in solvents, reagents, glassware, and other
sample-processing apparatus that lead to discrete artifacts or elevated baseline in liquid
chroinatograms. All reagents and apparatus must be routinely demonstrated to be free from
interferences under the conditions of the analysis by running laboratory reagent blanks as
described in Section 8.5.
3.1.1 Glassware must be scrupulously cleaned. 2 Clean all glassware as soon as possible after
use by thoroughly rinsing with the last solvent used in it. Follow by washing with hot
water and detergent and thorough rinsing with tap and reagent water. Drain dry, and
heat in an oven or muffle furnace at 400°C for 15 to 30 minutes. Do not heat volumetric
ware. Some thermally stable materials, such as PCBs, may not be eliminated by this
treatment. Thorough rinsing with acetone and pesticide-quality hexane may be substituted
for the heating. After drying and cooling, seal and store glassware in a clean
environment to prevent any accumulation of dust or other contaminants. Store inverted
or capped with aluminum foil.
3.1.2 The use of high-purity reagents and solvents helps to minimize interference problems.
Purification of solvents by distillation in all-glass systems may be required.
3.2 Matrix interferences may be caused by contaminants that are coextracted from the sample. The
extent of matrix interferences will vary considerably from source to source, depending upon the
nature and diversity of the industrial complex or municipality sampled. The cleanup procedure
in Section 11 can be used to overcome many of these interferences, but unique samples may
require additional cleanup approaches to achieve the MDL listed in Table 2.
4 SAFETY
4.1 The toxicity or carcinogemcity of each reagent used in this method has not been precisely defined;
however, each chemical compound should be treated as a potential health hazard. From this
viewpoint, exposure to these chemicals must be reduced to the lowest possible level by whatever
means available. The laboratory is responsible for maintaining a current awareness file of OSHA
regulations regarding the safe handling of the chemicals specified in this method. A reference file
of material data handling sheets should also be made available to all personnel involved in the
chemical analysis. Additional references to laboratory safety are available and have been
identified for the information of the analyst.
5. APPARA TUS AND MA TEPJALS
5.1 Sampling equipment, for discrete or composite sampling.
5.1.1 Grab-sample bottle: Amber borosiicate or flint glass, 1-L or 1-quart volume, fitted with
screw-caps lined with PTFE. Foil may be substituted for PTFE if the sample is not
corrosive. If amber bottles are not available, protect samples from light. The container
and cap liner must be washed, rinsed with acetone or methylene chloride, and dried
before use to minimize contamination.
5.1.2 Automatic sampler (optional): Must incorporate glass sample containers for the
collection of a minimum of 250 mL. Sample containers must be kept refrigerated at 4°C
528
-------
Method 640
and protected from light during compositing. If the sampler uses a peristaltic pump, a
minimum length of compressible silicone rubber tubing may be used. Before use,
however, the compressible tubing should be thoroughly rinsed with methanol, followed
by repeated rinsings with distilled water to minimize the potential for contamination of
the sample. An integrating flow meter is required to collect flow-proportional
composites.
5.2 Glassware. (All specifications are suggested. Catalog numbers are included for illustration only.)
5.2.1 Separatory funnel: 2000-mL, with PTFE stopcock.
5.2.2 Drying column: Chromatographic column 400 mm long by 10 mm ID with coarse fit.
5.2.3 Chromatographic column: 400 mm long by 19 mm ID with 250-mL reservoir at the top
and PTFE stopcock (Kontes K-420290 or equivalent).
5.2.4 Concentrator tube, Kuderna-Danish: 10-mL, graduated (Kontes K-570050-1025 or
equivalent). Calibration must be checked at the volumes employed in the test. A
ground-glass stopper is used to prevent evaporation of extracts.
5.2.5 Evaporative flask, Kuderna-Danish: 500-mL (Kontes K-570001-0500 or equivalent).
Attach to concentrator tube with springs.
5.2.6 Snyder column, Kuderna-Danish: Three-ball macro (Kontes K-503000-0121 or
equivalent).
5.2.7 Snyder column, Kuderna-Danish: Two-ball micro (Kontes K-569001-0219 or
equivalent).
5.2.8 Vials: Amber glass, 10- to 15-mL capacity with PTFE-lined screw-cap.
5.2.9 Erlenmeyer flask: 250-mL.
5.2.10 Graduated cylinder: 1000-mL.
5.2.11 Volumetric flask: 5-mL, 10-mL.
5.3 Boiling chips: Approximately 10/40 mesh carborundum. Heat at 400°C for 4 hours or perform
a Soxhiet extraction with methylene chloride.
5.4 Water bath: Heated, capable of temperature control (±2°C). The bath should be used in a hood.
5.5 Balance: Analytical, capable of accurately weighing to the nearest 0.0001 g.
5.6 Liquid chromatograph: Analytical system complete with liquid chromatograph suitable for
on-column injection and all required accessories including syringes, analytical columns, detectors,
and strip-chart recorder. A data system is recommended for measuring peak areas.
5.6.1 Column 1: Spherisorb-ODS, 5 ’ , 250 mm long by 4.6 mm ID or equivalent. This
column was used to develop the method performance statements in Section 14.
Alternative columns may be used in accordance with the provisions described in
Section 12.1.
5.6.2 Column 2: Lichrosorb RP-2, 5j , 250 mm long by 4.6 mm ID or equivalent.
529
-------
Method 640
6. REAGENTS
6.1 Reagent water: Reagent water is defined as a water in which an interferent is not observed at the
method detection limit of each parameter of interest.
6.2 Methylene chloride, methanol, acetonitrile, ethyl ether, and acetone: Distilled-in-glass quality or
equivalent. Ethyl ether must be free of peroxides as indicated by EM Quant test strips (available
from Scientific Products Co., Catalog No. P1126-8 and other suppliers). Procedures
recommended fOr removal of peroxides are provided with the test strips.
6.3 Sodium sulfate: ACS, granular, anhydrous; heated in a muffle furnace at 400°C overnight.
6.4 Silica gel: Davison Grade 923, 100-200 mesh, dried for 12 hours at 150°C.
6.5 IN sodium hydroxide: Dissolve 4 g of sodium hydroxide in 100 mL of distilled water.
* 6.6 iN sulfuric acid: Slowly add 2.8 mL of concentrated 112504 (94%) to about 50 mL of distilled
water DdutetoI00mLwithd stzlledwater
6.7 Sodium phosphate: inonobasic, ACS grade.
6.8 Sodium phosphate: dibasic, ACS grade.
6.9 Stock standard solutions (1.00 p.g4iL): Stock standard solutions can be prepared from pure
standard materials or purchased as certified solutions.
6.9.1 Prepare stock standard solutions by accurately weighing about 0.0100 g of pure material.
Dissolve the material m distilled-in-glass quality methanol and dilute to volume in a
10-mL volumetric flask. Larger volumes can be used at the convenience of the analyst.
If compound purity is certified at 96% or greater, the weight can be used without
correction to calculate the concentration of the stock standard. Commercially prepared
stock standards can be used at any concentration if they are certified by the manufacturer
or by an independent source.
6.9.2 Transfer the stock standard solutions into FFFE-sealed screw-cap bottles. Store at 40°C
and protect from light. Frequently check standard solutions for signs of degradation or
evaporation, especially just prior to preparing calibration standards from them.
6.9.3 Stock standard solutions must be replaced after 6 months, or sooner if comparison with
check standards indicates a problem.
7. CJ$J.I&9 41T1ON
7.1 Establish liquid chromatographic operating parameters equivalent to those indicated in Table 1.
The liquid chromatographic system can be calibrated using the external standard technique
(Section 7.2) or the internal standard technique (Section 7.3).
7.2 External standard calibration procedure.
7.2.1 For each compound of interest, prepare calibration standards at a minimum of three
concentration levels by adding volumes of one or more stock standards to a volumetric
flask and diluting to volume with methanol.. One of the external standards should be at
a concentration near, but above, the method detection limit. The other concentrations
should correspond to the range of concentrations expected in the sample concentrates or
should define the working range of the detector.
530
-------
Method 640
7.2.2 Using injection of 5 to 20 1 iL of each calibration standard, tabulate peak height or area
responses against the mass injected. The results can be used to prepare a calibration
curve for each compound. Alternatively, the ratio of the response to the mass injected,
defined as the calibration factor (CF), can be calculated for each compound at each
standard concentration. If the relative standard deviation of the calibration factor is less
than 10% over the working range, the average calibration factor can be used in place of
a calibration curve.
7.2.3 The working calibration curve or calibration factor must be verified on each working
shift by the measurement of one or more calibration standards. If the response for any
compound varies from the predicted response by more than ±10%, the test must be
repeated using a fresh calibration standard. Alternatively, a new calibration curve or
calibration factor must be prepared for that compound.
7.3 Internal standard calibration procedure: To use this approach, the analyst must select one or more
internal standards similar in analytical behavior to the compounds of interest. The analyst must
further demonstrate that the measurement of the internal standard is not affected by method or
matrix interferences. Due to these limitations, no internal standard applicable to all samples can
be suggested.
7.3.1
Prepare calibration standards at a minimum of three concentration levels for each
compound of interest by adding volumes of one or more stock standards to a volumetric
flask. To each calibration standard, add a known constant amount of one or more
internal standards, and dilute to volume with methanol. One of the standards should be
at a concentration near, but above, the method detection limit. The other concentrations
should correspond to the range of concentrations expected in the sample concentrates or
should define the working range of the detector.
7.3.2 Using injections of 5 to 20 iiL of each calibration standard, tabulate the peak height or
area responses against the concentration for each compound and internal standard.
Calculate response factors (RF) for each compound as follows:
Equation 1
= (A,)(C ,)
(A 1 , )(C,)
where
A, =
A 1 , =
C 1 =
C, =
Response for
Response for
Concentration
Concentration
the parameter to be measured
the internal standard
of the internal standard, in gIL
of the parameter to be measured, in gIL
If the RF value over the working range is constant, less than 10% relative standard
deviation, the RF can be assumed to be invariant and the average RF can be used for
calculations. Alternatively, the results can be used to plot a calibration curve of response
ratios, A /A 1 against RF.
531
-------
Method 640
7.3.3 The working calibration curve or RF must be verified on each working shift by the
measurement of one or more calibration standards. If the response for any compound
varies from the predicted response by more than ± 10%, the test must be repeated using
a fresh calibration standard. Alternatively, a new calibration curve must be prepared for
that compound.
7.4 Before using any cleanup procedure, the analyst must process a series of calibration
standards through the procedure to validate elution patterns and the absence of
interferences from there agents.
8. Qwuny CONTROL
8.1 Each laboratory using this method is required to operate a formal quality control program. The
minimum requirements of this program consist of an initial demonstration of laboratory capability
and the analysis of spiked samples as a continuing check on performance. The laboratory is
required to maintain performance records to define the quality of data that is generated.
8.1.1 Before performing any analyses, the analyst must demonstrate the ability to generate
acceptable accuracy and precision with this method. This ability is established as
described in Section 8.2.
8.1.2 In recognition of the rapid advances occurring in chromatography, the analyst is
permitted certain options to improve the separations or lower the cost of measurements.
Each time such modifications to the method are made, the analyst is required to repeat
the procedure in Section 8.2.
8.1.3 The laboratory must spike and analyze a minimum of 10% of all samples to monitor
continuing laboratory performance. This procedure is described in Section 8.4.
8.2 To establish the ability to generate acceptable accuracy and precision, the analyst must perform
the following operations.
8.2.1 Select a representative spike concentration for each compound to be measured. Using
stock standards, prepare a quality control check sample concentrate in methanol, 1000
times more concentrated than the selected concentrations.
8.2.2 Using a pipette, add 1.00 mL of the check sample concentrate to each of a minimum of
four 1000-mL aliquots of reagent water. A representative wastewater may be used in
place of the reagent water, but one or more additional aliquots must be analyzed to
determine background levels, and the spike level must exceed twice the background level
for the test to be valid. Analyze the aliquots according to the method beginning in
Section 10.
8.2.3 Calculate the average percent recovery (R), and the standard deviation of the percent
recovery (s), for the results. Wastewater background corrections must be made before
R and a calculations are performed.
8.2.4 Using the appropriate data from Table 2, determine the recovery and single-operator
precision expected for the method, and compare these results to the values measured in
Section 8.2.3. If the data are not comparable, the analyst must review potential problem
areas and repeat the test.
532
-------
Method 640
8.3 The analyst must calculate method performance criteria and define the performance of the
laboratory for each spike concentration and parameter being measured.
8.3.1 Calculate upper and lower control limits for method performance as follows:
Upper Control Limit (UCL) = R + 3s
Lower Control Limit (LCL) = R - 3s
where R and s are calculated as m Section 8.2.3. The UCL and LCL can be used to
construct control charts 6 that are useful in observing trends in performance.
8.3.2 The laboratory must develop and maintain separate accuracy statements of laboratory
performance for wastewater samples. An accuracy statement for the method is defined
as R ± s. The accuracy statement should be developed by the analysis of four aliquots
of wastewater as described in Section 8.2.2, followed by the calculation of R and s..
Alternatively, the analyst may use four wastewater data points gathered through the
requirement for continuing quality control in Section 8.4. The accuracy statements
should be updated regularly. 6
8.4 The laboratory is required to collect in duplicate a portion of their samples to monitor spike
recoveries. The frequency of spiked sample analysis must be at least 10% of all samples or one
sample per month, whichever is greater. One aliquot of the sample must be spiked and analyzed
as described in Section 8.2. If the recovery for a particular compound does not fall within the
control limits for method performance, the results reported for that compound in all samples
processed as part of the same set must be qualified as described in Section 13.3. The laboratory
should monitor the frequency of data so qualified to ensure that it remains at or below 5%.
8.5 Before processing any samples, the analyst should demonstrate though the analysis of a 1-L aliquot
of reagent water that all glassware and reagent interferences are under control. Each time a set
of samples is extracted or there is a change in reagents, a laboratory reagent blank should be
processed as a safeguard against laboratory contamination.
8.6 It is recommended that the laboratory adopt additional quality assurance practices for use with this
method. The specific practices that are most productive depend upon the needs of the laboratory
and the nature of the samples. Field duplicates may be analyzed to monitor the precision of the
sampling technique. When doubt exists over the identification of a peak on the chromatograni,
confirmatory techniques such as gas-chromatography with a dissimilar column, specific element
detector, or mass spectrometer must be used. Whenever possible, the laboratory should perform
analysis of standard reference materials and participate in relevant performance evaluation studies.
9. SAMPLES COLLECTiON, PRESERVA VON, AND HANDLING
9.1 Grab samples must be collected in glass containers. Conventional sampling practices 7 should be
followed; however, the bottle must not be prerinsed with sample before collection. Composite
samples should be collected in refrigerated glass containers in accordance with the requirements
of the program. Automatic sampling equipment must be as free as possible of plastic and other
potential sources of contamination.
9.2 The samples must be iced or refrigerated at 4°C from the time of collection until extraction.
533
-------
Method 640
9.3 Adjust the pH of the sample to 6 to 8 with sodium hydroxide or sulfuric acid immediately after
sampling.
10. SAMPLE EXrnAcTTON
10.1 Mark the water meniscus on the side of the sample bottle for later determination of sample
volume. Pour the entire sample into a 2-L separatory funnel. Check the pH of the sample with
wide range pH paper and adjust to 6 to 8 with iN sodium hydroxide or iN sulfuric acid.
Dissolve 5 g of monobasic sodium phosphate and 5 g of dibasic sodium phosphate in the sample.
10.2 Add 60 mL of methylene chloride to the sample bottle, seal, and shake 30 seconds to rinse the
inner walls. Transfer the solvent to the separatory funnel and extract the sample by shaking the
funnel for two minutes with periodic venting to release excess pressure. Allow the organic layer
to separate from the water phase for a minimum of 10 minutes. If the emulsion interface between
layers is more than one-third the volume of the solvent layer, the analyst must employ mechanical
techniques to complete the phase separation. The optimum technique depends upon the sample,
but may include stirring, filtration of the emulsion through glass wool, centrifugation, or other
physical methods. Collect the methylene chloride extract in a 250-mL Erlenmeyer flask.
10.3 Add a second 60-mL volume of methylene chloride to the sample bottle and repeat the extraction
procedure a second time, combining the extracts in the Erlenmeyer flask. Perform a third
extraction in the same manner.
10.4 Assemble a Kuderna-Danish (K-D) concentrator by attaching a 25-mL concentrator tube to a
500-mL evaporative flask. Other concentration devices or techniques may be used in place of the
K-D if the requirements of Section 8.2 are met.
10.5 Pour the combined extract through a drying column containing about 10 cm of anhydrous sodium
sulfate, and collect the extract in the K-D concentrator. Rinse the Erlenmeyer flask and column
with 20 to 30 mL of methylene chloride to complete the quantitative transfer. Once the flask rinse
has passed through the drying column, rinse the column with 30 to 40 mL of methylene chloride.
10.6 Add one or two clean boiling chips to the evaporative flask and attach a three-ball Snyder column.
Prewet the Snyder column by adding about 1 mL of methylene chloride to the top. Place the K-D
apparatus on a hot water bath, 60 to 65°C, so that the concentrator tube is partially immersed in
the hot water, and the entire lower rounded surface of the flask is bathed with hot vapor. Adjust
the vertical position of the apparatus and the water temperature as required to complete the
concentration in 15 to 20 minutes. Ax the proper rate of distillation, the balls of the column will
actively chatter but the chambers will not flood with condensed solvent. When the apparent
volume of liquid reaches 1 mL, remove the K-D apparatus and allow it to drain and cool for at
least 10 minutes.
10.7 Remove the macro-Snyder column and rinse the flask and its lower joint into the concentrator tube
with 1 to 2 mL of methylene chloride. Add one or two clean boiling chips and attach a two-ball
micro-Snyder column to the concentrator tube. Prewet the micro-Snyder column with methylene
chloride and concentrate the solvent extract as before. When an apparent volume of 0.5 mL is
reached, or the solution stops boiling, remove the K-D apparatus and allow it to drain and cool
for 10 minutes.
534
-------
Method 640
10.8 Remove the micro-Snyder column and adjust the volume of the extract to 1.0 mL with methylene
chloride. Stopper the concentrator tube and store refrigerated if further processing will not be
performed immediately. If the extract is to be stored longer than 2 days, transfer the extract to
a screw-capped vial with a PTFE-lined cap. If the sample extract requires no further cleanup,
proceed with Section 10.9. If the sample requires cleanup, proceed to Section 11.
10.9 Add one or two clean boiling chips to the concentrator tube along with l0-mL of methanol.
Attach a two-ball micro-Snyder column and prewet the micro-Snyder column with about 1 mL of
methanol. Concentrate the solvent extract as before to an apparent volume of 2 mL and allow it
to drain and cool for 10 minutes. Transfer the solvent extract to a 5 mL volumetric flask and
dilute to the mark with methanol. Proceed with the liquid chromatographic analysis in Section 12.
10.10 Determine the original sample volume by refilling the sample bottle to the mark and transferring
the water to a 1000-mL graduated cylinder. Record the sample volume to the nearest 5 mL.
11. CLEANUP AND SEPARA TJON
11.1 Cleanup procedures may not be necessary for a relatively clean sample matrix. The cleanup
procedure recommended in this method has been used for the analysis of various clean waters and
industrial effluents. If particular circumstances demand the use of an alternative cleanup
procedure, the analyst must determine the elution profile and demonstrate that the recovery of
each compound of interest is not less than 85%.
11.2 The following silica gel column cleanup procedure has been demonstrated to be applicable to
mercaptobenzothiazole.
11.2.1 Add lOg of silica gel to 100 mL of ethyl ether and 600 1 iL of reagent water in a 250-mL
Erlenmeyer flask. Shake vigorously for 15 minutes. Transfer the slurry to a
chromatographic column (silica gel may be retained with a plug of glass wool). Allow
the solvent to elute from the column until the silica gel is almost exposed to the air.
Wash the column with 100 mL of methylene chloride. Use a column flow of 2 to
2.5 mL/min throughout the wash and elution profiles.
11.2.2 Quantitatively add the sample extract from Section 10.8 to the head of the column.
Allow the solvent to elute from the column until the silica gel is almost exposed to the
air. Elute the column with 50 mL of methylene chloride. Discard this fraction.
11.2.3 Elute the column with 50 mL of 6% acetone in methylene chloride and collect eluate in
a K-D apparatus. Concentrate this fraction to 1 mL as described in Sections 10.6
and 10.7. Exchange solvent with methanol as described in Section 10.9 and proceed with
liquid chromatographic analysis.
12. LIQuID CHROMA TOGR4PHY
12.1 Table 1 summarizes the recommended operating conditions for the liquid chromatograph.
Included in this table are estimated retention times and method detection limits that can be
achieved by this method. Examples of the separations achieved by Column 1 and Column 2 are
shown in Figures 1 and 2. Other columns, chromatographic conditions, or detectors may be used
if the requirements of Section 8.2 are met.
12.2 Calibrate the liquid chromatographic system daily as described in Section 7.
535
-------
Method 640
12.3 If the internal standard approach is being used, the analyst must not add the internal standard to
the sample extracts until immediately before injection into the instrument. Mix thoroughly.
12.4 Inject 5 to 20 pL of the sample extract. Record the resulting peak sizes in area or peak height
units.
12.5 The width of the retention-time window used to make identifications should be based upon
measurements of actual retention-time variations of standards over the course of a day. Three
times the standard deviation of a retention time for a compound can be used to calculate a
suggested window size; however, the experience of the analyst should weigh heavily in the
interpretation of chromatograms.
12.6 If the response for the peak exceeds the working range of the system, dilute the extract and
lyze.
12.7 If the measurement of the peak response is prevented by the presence of interferences, further
cleanup is required.
13. CALCULATIONS
13.1 Determine the concentration of individual compounds in the sample.
13.1.1 If the external standard calibration procedure is used, calculate the amount of material
injected from the peak response using the calibration curve or calibration factor in Section
7.2.2. The concentration in the sample can be calculated as follows:
Equation 2
Concentration, igIL = _____
(V j)( r
isI ere
A = Amount of material injected, in ng
V 1 = Volume of extract injected, in p ..L
= Volume of total extract, in pL
= Volume of water extracted, in niL
13.1.2 If the internal standard calibration procedure was used, calculate the concentration in the
sample using the response factor (RF) determined in Section 7.3.2 as follows:
536
-------
Method 640
Equation 3
(A )(I )
Concentration, jigiL = (A ,) (RF) (V 0 )
where
= Response for parameter to be measured
A 1 , = Response for the internal standard
I , = Amount of internal standard added to each extract,
in
g
= Volume of water extracted, in L
13.2 Report results in micrograms per liter without correction for recovery data. When duplicate and
spiked samples are analyzed, report all data obtained with the sample results.
13.3 For samples processed as part of a set where the laboratory spiked sample recovery falls outside
of the control limits in Section 8.3, data for the affected compounds must be labeled as suspect.
14. METHOD PERFORMANCE
14.1 The method detection limit (MDL) is defined as the minimum concentration of a substance that
can be measured and reported with 99% confidence that the value is above zero. 8 The MDL
concentrations listed in Table I were obtained using reagent water. 1 Similar results were achieved
using representative wastewaters.
14.2 This method has been tested for linearity of recovery from spiked reagent water and has been
demonstrated to be applicable over the concentration range from 5 to 1000 gIL.
14.3 In a single laboratory, Battelle Columbus Laboratories, using spiked wastewater samples, the
average recoveries presented in Table 2 were obtained after silica gel cleanup. Seven replicates
of each of two different wastewaters were spiked and analyzed. The standard deviation of the
percent recovery is also included in Table 2.1
537
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Method 640
References
1. “Development of Methods for Pesticides in Wastewaters,” EPA Contract Report 68-03-2956 (In
preparation).
2. ASTM Annual Book of Standards, Part 31, D3694, “Standard Practice for Preparation of Sample
Containers and for Preservation,” American Society for Testing and Materials, Philadelphia,
Pennsylvania, p. 679, 1980.
3. “Carcinogens - Working with Carcinogens,” Department of Health, Education, and Welfare,
Public Health Service, Center for Disease Control, National Institute for Occupational Safety and
Health, Publication No. 77-206, August, 1977.
4. “OSHA Safety and Health Standards, General Industry,” (29 CFR 1910), Occupational Safety and
Health Administration, OSHA 2206 (Revised, January 1976).
5. “Safety in Academic Chemistry Laboratories,” American Chemical Society Publication,
Committee on Chemical Safety, 3rd Edition, 1979.
6. “Handbook for Analytical Quality Control in Water and Wastewater Laboratories,”
EPA-600/4-79-019, U.S. Environmental Protection Agency, Environmental Monitoring and
Support Laboratory - Cincinnati, Ohio, March 1979.
7. ASTM Annual Book of Standards, Part 31, D3370, “Standard Practice for Sampling Water,”
American Society for Testing and Materials, Philadelphia, Pennsylvania, p. 76, 1980.
8. Glaser, J.A., et. aL, “Trace Analysis for Wastewaters,” Environmental Science and Technology,
15, 1426 (1981).
538
-------
Method 640
Table 1. Chromatographic Conditions and Method Detection Limits
Retention Time tm/n ) MDL
Parameter Column 1 Column 2 (pg/Li
Mercaptobenzotbiazole 8.4 9.5 1.7
Column 1 conditions: Spherisorb-ODS, 5 &, 250mm long by 4.6 mm ID; 1 mL/min flow; 50/50
acetonitrile/water.
Column 2 conditions: Lichrosorb RP-2, 5 p, 250mm long by 4.6 mm ID; l mL/min flow; 10190
acetonitrilelwater.
Table 2. Single-Laboratory Accuracy and ‘Precision (a)
Average Standard Number
Sample Background Spike Recovery Deviation of
Parameter Type fbi (pg/Li (ci (pg/Li (96) (96) Replicates
Mercaptobenzo- 1 ND ‘5 79 5 7
thiazole -
1 ND 10 87 4 7
(a) Column 1 conditions were used.
(b) 1 = Municipal sewage effluent
(c) ND = Not detected
539
-------
Method 640
I I I I I I I I I I I I I I I I I I
0 1.5 3.0 4.5 6.0 7.5 9.0 10.5 12.0 13.5 15.0
Retention Time (minutes)
A -O 3O
Figure 1. HPLC-UV Chromatogram of 10 ng of Mercaptobenzothiazole (Column 1).
-------
MethOd 640
Mercaptobenzothiazole
/
J__I I I I I I I I I I I I I I I I
0 2.0 4.0 6.0 8.0 10.0 12.0 14.0 16.0 18.0 20.0
Retention Time (minutes)
A52-002-31
Figure 2. HPLC-UV Chromatogram of 10 ng of MercaptobenzOthiaZOle (Co’umn 2).
541
-------
Method 641
The Determination of
Thiabendazole in Municipal
and Industrial Waste water
-------
Method 641:
The Determination of Thiabendazole in Municipal and
Industrial Waste water
SCOPE AND APPliCATION
1.1 This method covers the determination of thiabendazole in municipal and industrial wastewater.
Parameter CAS No.
Thiabendazole 148 -79-8
1.2 The estimated detection limit (EDL) for thiabendazole is listed in Table 1. The EDL was
calculated from the minimum detectable response being equal to 5 times the background noise
using a 100-,LL injection. The EDL for a specific wastewater may be different depending on the
nature of interferences in the sample matrix.
1.3 This is a liquid chromatographic method applicable to the determination of thiabendazole in
municipal and industrial discharges. When this method is used to analyze unfamiliar samples for
thiabendazole, compound identification should be supported by at least one additional qualitative
technique.
1.4 This method is restricted to use by or under the supervision of analysts experienced in the
operation of liquid chromatographs and in the interpretation of chromatograms. Each analyst must
demonstrate the ability to generate acceptable results with this method using the procedure
described in Sections 9.2 and 9.3.
2. SUMMARY OF METHOD
2.1 Thiabendazole is analyzed in the sample matrix after solubiization with acid and filtration to
remove particulate matter. Chromatographic conditions are described which permit the separation
and accurate measurement of thiabendazole by direct aqueous injection and HPLC with
fluorescence detection.
3. INTERFERENCES
3.1 Solvent, reagents, glassware, and other sample-processing hardware may yield discrete artifacts
and/or elevated baselines causing misinterpretation of the chromatograms. All of these materials
must be demonstrated to be free from interferences under the conditions of the analysis by running
laboratory reagent blanks as described in Section 9.1.
3.1.1 The use of high-purity reagents and solvents helps to minimize interference problems.
Purification of solvents by distillation in all-glass systems may be required.
3.1.2 Glassware must be scrupulously cleaned. 1 Clean all glassware as soon as possible after
use by rinsing with the last solvent used in it. This should be followed by detergent
washing with hot water and rinses with tap water and reagent water. It should then be
drained dry and heated in a muffle furnace at 400°C for 15 to 30 minutes. Solvent rinses
545
-------
Method 641
with acetone and pesticide-quality hexane may be substituted for the heating. Volumetric
ware should not be heated in a muffle furnace. After drying and cooling, glassware
should be sealed and stored in a clean environment to prevent any accumulation of dust
or other contaminants. Store the glassware inverted or capped with aluminum foil.
3.2 Matrix interferences may be caused by fluorescing contaminants that coelute with thiabendazole.
The extent of matrix interferences will vary considerably from source to source, depending upon
the nature and diversity of the industrial complex or municipality being sampled. Matrix
interferences caused by the presence of particulate matter are removed by filtration. Unique
samples may require additional cleanup approaches to achieve the detection limit listed in Table 1.
4. SAFETY
4.1 The toxicity or carcinogenicity of each reagent used in this method has not been precisely defined;
however, each chemical compound should be treated as a potential health hazard. From this
viewpoint, exposure to these chemicals must be reduced to the lowest possible level by whatever
means available. The laboratory is responsible for maintaining a current awareness file of OSHA
regulations regarding the safe handling of the chemicals specified in this method. A reference file
of material data handling sheets should also be made available to all personnel involved in the
chemical analysis. Additional references to laboratory safety are available and have been
identifie& for the information of the analyst.
5. APPARA TUS AND EQUIPMENT
5.1 Sampling.equipment for discrete sampling.
5.1.1 Vial: 25-mL capacity or larger, equipped with a screw-cap with hole in center (Pierce
13074 or equivalent). Detergent wash, rinse with tap and distilled water, and dry at
105°C before use.
5.1.2 Vial: 3.5-niL, equipped with a screw-cap with hole in center (Pierce 13019 or
equivalent). Wash vial and cap as in Section 5.1.1.
5.1.3 Septum: PTFE-faced silicone (Pierce 12722 or equivalent). Detergent wash and dry at
105°C for 1 hour before use.
5.1.4 Septum: PTFE-faced silicone (Pierce 12712 or equivalent). Detergent wash and dry at
105° for 1 hour before use.
5.2 Syringe: Glass, 5-mL with Leur tip.
5.3 Syringe-filter holder: Stainless steel with Leur connection (Rainin 38 to 101 or equivalent).
5.4 Filters: 13 mm, Nylon 66, O. 4 5-p pore (Rainin 38 to 112 or equivalent).
5.5 Balance: Analytical, capable of accurately weighing to the nearest 0.000! g.
5.6 High performance liquid chromatography (HPLC) apparatus: Analytical system complete with
liquid chromatograph and all required accessories including syringes, analytical columns, and
mobile phases. The system must be compatible with the specified detectors and strip-chart
recorder. A data system is recommended for measuring peak areas.
5.6.1 Isocratic pumping system, constant flow.
5.6.2 Injector valve (Rheodyne 7125 or equivalent) with 100-giL loop.
546
-------
Method 641
5.6.3 Column: 250 mm long by 4.6 mm ID, stainless steel, packed with reverse-phase
Ultrasphere ODS, 10 .
5.6.4 Fluorescence detector, for excitation at 300 urn and emission at 360 nm (Perkin Elmer
650 to IS or equivalent). Fluorometer should have dispersive optics for excitation and
utilize either filter or dispersive optics at the emission detector.
5.6.5 Strip-chart recorder compatible with detector, 250 mm. (A data system for measuring
peak areas is recommended.)
6. REAGENTS AND CONSUMABLE MA TERIALS
6.1 Reagent water: Reagent water is defined as a water in which an interferent is not observed at the
EDL of each parameter of interest.
6.2 Sodium hydroxide solution (iON): Dissolve 40 grams of NaOH in reagent water and dilute to
100 mL.
6.3 Sodium thiosulfate: ACS, granular.
6.4 Sulfuric acid solution (1 ÷ 1): Slowly add 50 mL of H 2 S0 4 (specific gravity 1.84) to 50 mL of
reagent water.
6.5 HPLC buffer (pH 8.2): Add 8 mL of triethanolarnine (Eastman 1599) and 1 mL of glacial acetic
acid (ACS) to 1 L of reagent water.
6.6 High-purity methanol: HPLC quality, distilled in glass.
6.7 Stock standard solution (1.0 &g/ iL): Stock standard solutions are prepared from pure standard
material or purchased as a certified solution.
6.7.1
Prepare the stock standard solution by accurately weighing about 0.OlOOg of pure
material. Dissolve the material in pesticide-quality methanol, dilute to volume in a
10-niL volumetric flask. Larger volumes can be used at the convenience of the analyst.
When compound purity is certified at 96% or greater, the weight can be used without
correction to calculate the concentration of the stock standard. Commercially prepared
stock standards can be used at any concentration if they are certified by the manufacturer
or by an independent source.
6.7.2 Transfer the stock standard to a PTFE-sealed screw-cap bottle. Store at 4°C and protect
from light. The stock standard should be checked frequently for signs of degradation or
evaporation, especially just prior to preparing calibration standards.
6.7.3 The stock standard must be replaced after 6 months, or when comparison with quality
control check samples indicates a problem.
7. SAMPLE COLLECTION, PRESERVATION, AND STORAGE
7.1 Collect all samples in duplicate. Grab samples must be collected in glass containers.
Conventional sampling practices should be followed, except that the bottle must not be prewashed
with sample before collection.
7.2 The samples must be iced or refrigerated at 4°C from the time of collection until analysis.
Chemical preservatives should not be used in the field unless more than 24 hours will elapse
before delivery to the laboratory. If the samples will not be analyzed within 48 hours of
547
-------
Method 641
collection, the sample should be adjusted to a pH range of 1.0 to 3.0 with sodium hydroxide or
sulfuric acid, and 35 mg of sodium thiosulfate per liter of sample for each part per million of free
chlorine should be added.
7.3 All samples must be analyzed within 30 days of collection. 6
8. CAIJBRA flON AND STANDARIZA liON
8.1 Establish liquid chtomatographic operating parameters equivalent to those indicated in Table 1.
8.2 Prepare calibration standards at a minimum of three concentration levels of thiabendazole by
adding volumes of the stock standard to a volumetric flask and diluting to volume with HPLC
mobile phase. One of the standards should be at a concentration near, but greater than, the EDL,
and the other concentrations should correspond to the expected range of concentrations found in
real samples or should define the working range of the detector.
8.3 Using injections of 100 p L of each calibration standard, tabulate peak height or area responses
against the mass injected. The results are used to prepare a calibration curve for thiabendazole.
Alternatively, if the ratio of response to amount injected (calibration factor) is a constant over the
working range (<10% relative standard deviation), linearity of the calibration curve can be
assumed and the average ratio or calibration factor can be used in place of a calibration curve.
8.4 h e working calibration curve or calibration factor must be verified on each working day by the
measurement of one or more calibration standards. If the response for thiabendazole varies from
the predicted response by more than ± 10%, the test must be repeated using a fresh calibration
standard. Alternatively, a new calibration curve or factor must be prepared.
8.5 Befoi e using any cleanup procedure, the analyst must process a series of calibration standards
through the procedure to validate elution patterns and the absence of interferences from the
reagents.
9. Qawn’ CONTROL
9.1 Monitoring for interferences.
9.1.1 Analyze a laboratory reagent blank as described in Section 10 each time a set of samples
is extracted. A laboratory reagent blank is an aliquot of reagent water. If the reagent
blank contains a reportable level of thiabendazole, immediately check the entire analytical
system to locate and correct for possible interferences and repeat the test.
9.2 Assessing accuracy.
9.2.1 After every ten samples, and preferably in the middle of each day, analyze a laboratory
control standard. Calibration standards may not be used for accuracy assessments and
the laboratory control standard may not be used for calibration of the analytical system.
9.2.1.1 Laboratory control standard concentrate: From the stock standard prepared
as described in Section 6.7, prepare a laboratory control standard concentrate
that contains thiabendazole at a concentration of 2 jig/mL in methanol or
other suitable solvent. 7
548
-------
Method 641
9.2.1.2 Laboratory control standard: using a pipette or microliter syringe, add
50.0 1 iL of the laboratory control standard concentrate to a 10-mL aliquot of
reagent water contained in a 10-mL volumetric flask.
9.2.1.3 Analyze the laboratory control standard as described in Section 10. Calculate
the percent recovery (P 1 ) with the equation:
Equation 1
p
where
S = the analytical results from the laboratory control standard, in ig/L
= the A7zo z concentration of the spike, in ig/L
9.2.2 At least annually, the laboratory should participate in formal performance evaluation
studies, where solutions of unknown concentrations are analyzed and the performance of
all participants is compared.
9.3
Assessing precision.
9.3.1 Precision assessments for this method are based upon the analysis of field duplicates
(Section 7.1). Analyze both sample vials for at least 10% of all samples. To the extent
practical, the samples for duplication should contain reportable levels of thiabendazole.
9.3.2 Calculate the relative range (RR) with the equation:
Equation 2
100R
RR.=
‘x i
where
R= the absolute difference between the duplicate measurements X 1 and X 2 , in gIL
x+;
X,= the average concentration found in pg/L
9.3.3 Individual relative range measurements are pooled to determine average relative range
or to develop an expression of relative range as a function of concentration.
10. PROCEDURE
10.1 Sample preparation.
10.1.1 Adjust the pH of the sample to pH 1 to 3 with sulfuric acid solution.
10.1.2 Assemble the syringe-filtration assembly by attaching the filter holder (with filter) to a
5-mL glass syringe equipped with a Leur tip.
10.1.3 Remove the barrel from the syringe and pour a 4- to 5-mL aliquot of the acidifield
sample into the syringe, allowing room for reinsertion of the syringe barrel.
549
-------
Method 641
10.1.4 Filter a portion of the sample through 0.45-si filter using a syringe-filter holder. The
first few milliliters should be discarded. Collect the filtrate in a 4-mL vial equipped with
a PTFE-sealed screw-cap.
10.1.5 The syringe and filter holder should be rinsed with acetone or methanol and then HPLC-
grade water between samples.
10.2 Cleanup and separation.
10.2.1 Cleanup procedures may not be necessary for a relatively clean sample matrix. Use of
fluorescent detectors, however, often obviates the necessity for cleanup of relatively clean
sample matrices. If particular circumstances demand the use of an alternative cleanup
procedure, the analyst must determine the elution profile and demonstrate that recovery
is no less than 85%.
10.3 Liquid chromatographic analysis.
10.3.1 Table I summarizes the recommended operating conditions for the liquid chromatograph.
Included in this table are the estimated retention time and estimated detection limit that
can be achieved by this method. An example chromatogram achieved by this column is
shown in Figure 1. Figure 2 is a chromatogram of thiabendazole in a POTW wastewater
sample. Other columns, chromatographic conditions, or detectors may be used if data
quality comparable to Table 2 is achieved.
10.3.2 Calibrate the system daily as described in Section 8.
10.4 Inject 100 L of the filtered aqueous sample. Monitor the column eluent at excitation wavelength
300 urn (5-nm slit width) and emission wavelength 360 nm (10-nm slit width). Record the
resulting peak size in area or peak height units.
10.5 The retention-time window used to make identifications should be based upon measurements of
actual retention-time variations of standards over the course of a day. Three times the standard
deviation of a retention time for a compound can be used to calculate a suggested window size;
however, the experience of the analyst should weigh heavily in the interpretation of
chromatograms.
10.6 If the response for the peak exceeds the working range of the system, dilute the sample with
mobile phase and reanalyze.
10.7 If the measurement of the peak response is prevented by the presence of interferences, further
cleanup is Tequired.
11. CALCULA TJONS
11.1 Determine the concentration of thiabendazole in the sample.
1 1.1.1 Calculate the amount of thiabendazole injected from the peak response using the
calibration curve or calibration factor in Section 8.2.2. The concentration in the sample
can be calculated from the following equation:
550
-------
Method 641
Equation 3
Concentration, g/L = ( A)(100 )
where
A = Amount of the rhiabendazole injected, in ng
V 1 = Volume of sample injected, in pL
11.2 Report results in micrograms per liter without correction for recovery data. When duplicate and
spiked samples are analyzed, report all data obtained with the sample results.
12. METHOD PERFORMANCE
12.1 The EDL and associated chromatographic conditions for thiabendazole are listed in Table i. The
EDL is defined as the minimum response being equal to 5 times the background noise, using a
lOO- L injection.
12.2 Single-operator accuracy and precision studies were conducted by Environmental Science and
Engineering, Inc ., 6 using a spiked PO1’W sample. The results of these studies are presented in
Table 2.
551
-------
Method 641
References
1. ASTM Annual Book of Standards, Part 31, ‘Standard Practice for Preparation of Sample
Containers and for Preservation,’ American Society for Testing and Materials, Philadelphia,
Pennsylvania, p. 679, 1980.
2. ‘Carcinogens - Working with Carcinogens,’ Department of Health, Education, and Welfare,
Public Health Service, Center for Disease Control, National Institute for Occupational Safety and
Health, Publication No. 77-206, Aug. 1977.
3. ‘OSHA Safety and Health Standards, General Industry’ (29 CFR 1910), Occupational Safety and
Health Administration, OSHA 2206 (Revised, January 1976).
4. ‘Safety in Academic Chemistry Laboratories,’ American Chemical Society Publication,
Committee on Chemical Safety, 3rd Edition, 1979.
5. ASTM Annual Book of Standards, Vol. 11.01, D3370, ‘ tmidard Practice for Sampling Water,’
American Society for Testing and Materials, Philadelphia, PA, 1986.
6. Test procedures for Pesticides in Wastewaters, EPA Contract Report 68-03-2897, unpublished
report available from U.S. Environmental Protection Agency, Environmental Monitoring and
Support Laboratory, Cincinnati, Ohio.
7. ‘Handbook for Analytical Quality Control in Water and Wastewater Laboratories,’
EPA-60014-79-019, U.S. Environmental Protection Agency, Environmental Monitoring and
Support Laboratory, Cincinnati, Ohio, March 1979.
8. ‘Evaluation of Ten Pesticides Methods,’ U.S. Environmental Protection Agency, Contract No.
68-03-1760, Task No. 11, U.S. Environmental Monitoring and Support Laboratory, Cincinnati,
Ohio.
552
-------
Method 641
Table 1. Liquid Chromatography of Thiabendazole*
Retention Time Estimated Detection
Compound (minI Limit (pg/LI
Thiabendazole 4.3 1.7
*IWLC conditions: 10 reverse-phase Ultrasphere ODS; Column: 250 mm long by 4.6 mm ID;
isocratic 70% methanoll30% buffer; flow rate 1 mL/min.
Table 2. Single-Operator Accuracy and Precision*
Parameter Spike Number of Average Standard
Concentration Replicates Percent Deviation (%)
(pg/LI Recovery
Thiabendazole 12.5 7 100 9.5
625 7 92.8 4.5
*p J Vj effluent was used in this study.
553
-------
Method 641
Thiabendazole
1.0 2.0 3.0 4.0 5.0 6.0
Retention Time (minutes)
Figure 1. HPLC of Thiabendazote
0
554
-------
Method 641
A52 8
Figure 2. Chromatogram of Thiabendazole in Wastewater Sample.
Thiabendazole
0 1.0 2.0 3.0 4.0 5.0 6.0
Retention Time (minutes)
555
-------
Method 642
The Determination of
Biphenyl and Ortho-
PhenyipheflOl in Municipal
and industrial Waste water
-------
Method 642
The Determination of Biphenyl and Ortho-Phenyiphenol in
Municipal and Industrial Waste water
1. SCOPE AND APPLiCATION
1.1 This method covers the determination of biphenyl and o-phenylphenol in municipal and industrial
wastewater.
Parameter CA S No.
Biphenyl 92-52-4
o-phenylphenol 132-27-4
1.2 The estimated detection limits (EDL) for the parameters above are listed in Table 1. The EDLs
were calculated from the minimum detectable response being equal to 5 times the background
noise using a 2-mi. final extract volume of a 1-L sample and an injection volume of 50 L. The
EDL for a specific wastewater may be different depending on the nature of interferences in the
sample matrix.
1.3 This is a high performance liquid chromatographic (HPLC) method applicable to the determination
of the compounds listed above in municipal and industrial discharges. When this method is used
to analyze unfamiliar samples for any or all of the compounds above, compound identification
should be supported by at least one additional qualitative technique. This method describes
analytical conditions for a second HPLC column that can be used to confirm measurements made
with the primary column.
1.4 This method is restricted to use by or under the supervision of analysts experienced in the
operation of liquid chromatographs and in the interpretation of liquid chromatograms.
2. SUMMARY OF METHOD
2.1 The fungicides are removed from the sample matrix by extraction with methylene chloride. The
extract is dried, exchanged to acetonitrile or methanol, and analyzed by liquid chromatography
with ultraviolet (UV) detection.
3. INTERFERENCES
3.1 Solvent, reagents, glassware, and other sample-processing hardware may yield discrete artifacts
and/or elevated baselines causing misinterpretation of liquid chromatograms. All of these
materials must be demonstrated to be free from interferences under the conditions of the analysis
by running laboratory reagent blanks as described in Section 9.1.
3.1.1 The use of high-purity reagents and solvents helps to minimize interference problems.
Purification of solvents by distillation in all-glass systems may be required.
559
-------
Mefhoo 642
3.1.2 Glassware must be scrupulously cleaned. 1 Clean all glassware as soon as possible after
use by rinsing with the last solvent used in it. This should be followed by detergent
washing with hot water and rinses with tap water and reagent water. It should then be
drained dry and heated in a muffle furnace at 400°C for 15 to 30 minutes. Solvent rinses
with acetone and pesticide-quality hexane may be substituted for the heating. Volumetric
ware should not be heated in a muffle furnace. After drying and cooling, glassware
should be sealed and stored in a clean environment to prevent any accumulation of dust
or other contaminants. Store the glassware inverted or capped with aluminum foil.
3.2 Matrix interferences may be caused by UV-active contaminants that are coextracted from the
samples. The extent of matrix interferences will vary considerably from source to source,
depending upon the nature and diversity of the industrial complex or municipality being sampled.
While general cleanup procedures are provided as part of this method, unique samples may
require additional cleanup approaches to achieve the detection limits listed in Table 1.
4. SAFETY
4.1 The toxicity or carcinogenicity of each reagent used in this method has not been precisely defined;
however, each chemical compound should be treated as a potential health hazard. From this
viewpoint, exposure to these chemicals must be reduced to the lowest possible level by whatever
means available. The laboratory is responsible for maintaining a current awareness file of OSHA
regulations regarding the safe handling of the chemicals specified in this method. A reference file
of material data handling sheets should also be made available to all personnel involved in the
chemical analysis. Additional references to laboratory safety are available and have been
identifled 2 fur the information of the analyst.
5. APPARA TUS AND EQUIPMENT
5.1 Sample containers: Narrow-mouth glass bottles, 1-L or 1-quart volume, equipped with
polytetrafluoroethylene (PTFE)-lined screw-caps. Wide-mouth glass bottles, 1-quart volume,
equipped with FITE-Iined screw-caps may also be used. Prior to use, wash bottles and cap liners
with detergent and rinse with tap and reagent water. Allow the bottles and cap liners to air dry,
then muffle the bottles at 400°C for 1 hour. After cooling, rinse the bottles and cap liners with
hexane, seal the bottles, and store in a dust-free environment.
5.1.1 Automatic sampler (optional): Must incorporate glass sample containers for the
collection of a minimum of 250 mL. Sample containers must be kept refrigerated at 4°C
and protected from light during compositing. If the sampler uses a peristaltic pump, a
minimum length of compressible silicone rubber tubing may be used. Before use,
however, the compressible tubing should be thoroughly rinsed with methanol, followed
by repeated rinsings with reagent water to minimize the potential for contamination of
the sample. An integrating flow meter is required to collect flow-proportional
composites.
5.2 Kuderna-Danish (K-D) glassware.
5.2.1 Snyder column, Kuderna-Danish: Three-ball macro (Kontes K-.503000-0121 or
equivalent) and two-ball micro (Kontes K-569001-0219 or equivalent).
560
-------
Method 642
5.2.2 Concentrator tube: 10-inL, graduated (Kontes K-5’70050-1025 or equivalent) with
ground-glass stopper.
5.2.3 Evaporative flask: 500-mL (Kontes K-570001-0500 or equivalent). Attach to concentra-
tor tube with springs.
5.3 High performance liquid chromatography (HPLC) apparatus: Analytical system complete with
liquid chromatograph and all required accessories including syringes, analytical columns, and
mobile phases. The system must be compatible with the specified detectors and strip-chart
recorder. A data system is recommended for measuring peak areas.
5.3.1 Gradient pumping system, constant flow.
5.3.2 Injector valve (Rheodyne 7125 or equivalent) with 50-FL loop.
5.3.3 Column 1: 250 mm long by 4.6 mm ID, stainless steel, packed with reverse-phase
Perkin Elmer HC-ODS Sil-X 10 , or equivalent.
5.3.4 Column 2: 250 mm long by 4.6 mm ID, packed with reverse-phase Dupont Zorbax
ODS, 6 to 7 i, or equivalent.
5.3.5 Ultraviolet detector, capable of monitoring at 254 mu.
5.3.6 Strip-chart recorder compatible with detector, 250 mm. (A data system for measuring
peak areas is recommended.)
5.4 Chromatographic column: 300 mm long by 10 mm ID Chromaflex, equipped with coarse-fitted
bottom plate and PTFE stopcock. (Kontes K-420540-0213 or equivalent).
5.5 Drying column: Approximately 400 mm long by 20 mm ID borosilicate glass, equipped with
coarse-fritted bottom plate.
5.6 Miscellaneous.
5.6.1 Balance: Analytical, capable of accurately weighing to the nearest 0.0001 g.
5.6.2 Separatory funnels: 2-L, 500-mL, and 250-mL, equipped with PTFE stopcocks.
5.6.3 Boiling chips: Approximately 10/40 mesh. Heat to 400°C for 30 minutes or perform
a Soxhiet extraction with methylene chloride for 2 hours.
5.6.4 Water bath: Heated with concentric ring cover, capable of temperature control (±2°C).
The bath should be used in a hood.
5.6.5 Standard solution storage containers: 15-mL bottles with PTFE-lined screw-caps.
6. REAGENTS AND CONSUMABLE MA TEPJALS
6.1 Reagents.
6.1.1 Acetone, acetonitrile, methanol, and methylene chloride: Demonstrated to be free of
analytes and interferences.
6.1.2 Reagent water: Reagent water is defined as a water in which an interferent is not
observed at the method detection limit of each parameter of interest. The water is held
at 90°C. Store in clean, narrow-mouth bottles with PTFE-lined septa and screw-caps.
6.1.3 Sodium sulfate: Granular, anhydrous. Condition by heating at 400°C for 4 hours in a
shallow tray.
56?
-------
Method 642
6.1.4 HPLC Mobile Phase 1: Add 400 mL of acetonitrile to a 1-L volumetric flask and dilute
to volume with reagent water.
6.1.5 HPLC Mobile Phase 2: Add 500 mL of methanol to a 1-L volumetric flask and dilute
to volume with reagent water.
6.2 Standard stock solutions (1.00 pg4iL): These solutions may be purchased as certified solutions
or prepared from pure standard materials using the following procedures.
6.2.1 Prepare Stock standard solutions by accurately weighing about 0.0100 g of pure material.
Dissolve the material in pesticide-quality methanol or acetonitrile, dilute to volume in a
10-mL volumetric flask. Larger volumes can be used at the convenience of the analyst.
When compound purity is certified at 96% or greater, the weight can be used without
correction to calculate the concentration of the stock standard. Commercially prepared
stock standards can be used at any concentration if they are certified by the manufacturer
or by an independent source.
6.2.2 Transfer the stock standards to PTFE-sealed screw-cap bottles. Store at 4°C and protect
from light. Stock standards should be checked frequently for signs of degradation or
evaporation, especially just prior to preparing calibration standards from them.
6.2.3 Stock standards must be replaced after 6 months, or when comparison with quality
control check samples indicates a problem.
7. SAMPLE COLLECTION, PRESERVATION, AND STORAGE
7.1 Collect all samples in duplicate. Grab samples must be collected in glass containers.
Conventional sampling practic& should be followed, except that the bottle must not be prewashed
with sample before collection.
7.2 The samples must be iced or refrigerated at 4°C from the time of collection until analysis.
7.3 Mi samples must be extracted and analyzed as soon as possible after sampling, since preservation
studies’ have shown that these compounds undergo almost complete decomposition within 7 days.
8. CAliBRATION
8.1 Establish liquid chromatographic operating parameters equivalent to those indicated in Table 1.
8.2 Prepare calibration standards at a minimum of three concentration levels of the analytes by adding
volumes of the stock standard to a volumetric flask and diluting to volume with HPLC mobile
phase (40% acetonitrile in water or 50% methanol in water). One of the standards should be at
a concentration near, but greater than, the EDL, and the other concentrations should correspond
to the expected range of concentrations found in real samples or should define the working range
of the detector.
8.3 Using injections of 50 1 iL of each calibration standard, tabulate peak height or area responses
against the mass injected. The results are used to prepare a calibration curve for the analytes.
Alternatively, if the ratio of response to amount injected (calibration factor) is a constant over the
working range (<10% relative standard deviation), linearity of the calibration curve can be
assumed and the average ratio or calibration factor can be used in place of a calibration curve.
562
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____________________________________________________ Method 642
8.4 The working calibration curve or calibration factor must be verified on each working day by the
measurement of one or more calibration standards. If the response for any analyte varies from
the predicted response by more than ±10%, the test must be repeated using a fresh calibration
standard. Alternatively, a new calibration curve or factor must be prepared.
8.5 Before using any cleanup procedure, the analyst must process a series of calibration standards
through the procedure to validate elution patterns and the absence of interferences from the
reagents.
9. QUALITY CONTROL
9.1 Monitoring for interferences: Analyze a laboratory reagent blank each time a set of samples is
extracted. A laboratory reagent blank is an aliquot of reagent water. If the reagent blank contains
a reportable level of the analytes, immediately check the entire analytical system to locate and
correct for possible interferences and repeat the test.
9.2 Assessing accuracy.
9.2.1 After every ten samples, and preferably in the middle of each day, analyze a laboratory
control standard. Calibration standards may not be used for accuracy assessments and
the laboratory control standard may not be used for calibration of the analytical system.
9.2.1.1 Laboratory control standard concentrate: From stock standards prepared as
described in Section 6.2, prepare a laboratory control standard concentrate
that contains the analytes at a concentration of 2 zg/mL in methanol or
acetonitrile.
9.2.1.2 Laboratory control standard: Using a pipette, add 1.0 mL of the laboratory
control standard concentrate to a 1-L aliquot of reagent water contained in a
1000-mL volumetric flask.
9.2.1.3 Analyze the laboratory control standard as described in Section 10. Calculate
the percent recovery (1’) with the equation:
Equation I
ics ,
where
= The analytical results from the laboratory control standard, in ,2gIL
7 ’ , = The bzown concentration of the spike, in gIL
9.2.2 At least annually, the laboratory should participate in formal performance evaluation
studies, where solutions of unknown concentrations are analyzed and the performance of
all participants is compared. 7
9.3 Assessing precision.
9.3.1 Precision assessments for this method are based upon the analysis of field duplicates
(Section. 7.1). Analyze both sample bottles for at least 10% of all samples. To the
563
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Method 642
extent practical, the samples for duplication should contain reportable levels of the
9.3.2 Calculate the relative range 7 (RR 1 ) with the equation:
Equation 2
= IOOR ,
where
= The absolute d rence between the duplicate measurements X 1 and X , in ig/L
I, = The awrage concentration found. 1 , in &gIL
9.3.3 Individual relative range measurements are pooled to determine average relative range
or to develop an expression of relative range as a function of concentration.
10. PROCEDURE
10.1 Sample extraction.
10.1.1 Mark the wat& meniscus on the side of the sample bottle for later determination of
sample volume. Pour the entire sample into a 2-L separatory funnel.
10.1.2 Add 60 mL of methylene chloride to the sample bottle and shake for 30 seconds to rinse
the walls. Transfer the solvent to the separatory funnel and extract the sample by shaking
the funnel for 2 minutes with periodic venting to release vapor pressure. Allow the
organic layer to separate from the water phase for a minimum of 10 minutes. If the
emulsion interface between layers is more than one-third the volume of the solvent layer,
the analyst must employ mechanical techniques to complete the phase separation. The
optimum technique depends on the sample, but may include stirring, filtration of the
emulsion through glass wool, or centrifugation. Collect the extract in a 250-mL
Erlenmeyer flask.
10.1.3 Add an additional 60-niL volume of methylene chloride to the sample bottle and complete
the extraction procedure a second time, combining the extracts in the Erlenmeyer flask.
10.1.4 Perform a third extraction in the same manner. Pour the combined extract through a
drying column containing about 10 cm of anhydrous sodium sulfate, collecting the extract
in a 500 mL K-D flask equipped with a 10-mL concentrator tube. Rinse the Erlenmeyer
flask and column with about 30 mL of methylene chloride to complete the transfer.
10.1.5 Add one or two clean boiling chips to the flask and attach a three-ball Snyder column.
Prewet the Snyder column by adding about 1 mL of methylene chloride to the top. Place
theK-Dapparatusonahotwaterbath(SOto 85°C) so thatthe concentratortube is
partiaHy immersed in the hot water and the entire lower rounded surface of the flask is
bathed in steam. Adjust the vertical position of the apparatus and the water temperature
as required to complete the concentration in 15 to 20 minutes. At the proper rate of
564
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Method 642
distillation, the bails of the column will actively chatter but the chambers will not flood.
When the apparent volume of liquid reaches 5 mL, remove the K-D apparatus and allow
it to drain and cool for at least 10 minutes. If the extract requires cleanup, proceed to
Section 10.2. If the extract does not require cleanup, proceed with Sections 10.1.6
and 10.1.7.
10.1.6 Add 50 mL of methanol or acetonitrile and a clean boiling chip to the flask and repeat
the concentration as described above. When the apparent volume of the liquid reaches
1 mL, remove the K-I) apparatus and allow it to drain and cool for at least 10 minutes.
Remove the Snyder column and rinse the flask and its lower joint into the concentrator
tube with 1 to 2 mL of methanol or acetonitrile. A 5-mL syringe is recommended for
this operation.
10.1.7 Add a clean boiling chip to the concentrator tube. Attach a two-ball micro-Snyder
column. Prewet the micro-Snyder column by adding about 0.5 mL of methanol or
acetomtrile to the top. Place the micro K-D apparatus on a hot water bath (80 to 85°C)
so that the concentrator tube is partially immersed in the hot water. Adjust the vertical
position of the apparatus and water temperature as required to complete the concentration
in 5 to 10 minutes. At the proper rate of distillation, the balls will actively chatter but
the chambers will not flood. When the apparent volume of liquid reaches 0.5 mJ .,
remove the K-D apparatus and allow it to drain and cool for at least 10 minutes.
Remove the micro-Snyder column and rinse its lower joint into the concentrator tube with
a small volume of methanol or acetonitrile. Adjust the volume to 1.0 mL with methanol
or acetonitrile. Add 1.0 mL of reagent water to the extract if methanol or 1.5 mL of
reagent water to the extract if acetonitrile (Table 1).
NOTE: At high concentrations (approximately 1,(XY) mg/L or greater) of biphenyl in the
extract, low recoveries may be obtained due to insolubility in the acetonitrile. Larger
volumes of acetonitrile or acetone may be required to dissolve all the biphenyl and to
prevent preØpitation.
10.1.8 Determine the original sample volume by refilling the sample bottle to the mark and
transferring the liquid to a I ,000-mL graduated cylinder. Record the sample volume to
the nearest 5 mL.
10.2 Cleanup and separation.
10.2.1 Cleanup procedures may not be necessary for a relatively clean sample matrix. If
particular circumstances demand the use of a cleanup procedure, the analyst must
determine the elution profile and demonstrate that the recovery of each compound of
interest is no less than 85%.
10.2.2 Prior to HPLC analysis, the composition of the extracts must be as specified under
chromatographic conditions in Table 1 and described in Sections 10.1.6 and 10.1.7.
10.2.3 Proceed with liquid chromatography as described in Section 10.3.
10.3 Liquid chromatography analysis.
10.3.1 Table 1 summarizes the recommended operating conditions for the liquid chromatograph.
Included in this table are the estimated retention times and estimated detection limits that
565
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Method 642
can be achieved by this method. An example of the separation achieved by Column 1
of the analytas in a POTW extract is shown in Figure 1. Other columns,
chromatographic conditions,’ or detectors may be used if data quality comparable to
Table 2 is achieved.
10.3.2 Calibrate the system daily as described in Section 8.
10.3.3 Inject 50 iL of the sample extract. Monitor the column eluent at 254 nm. Record the
resulting peak size in area or peak height units.
10.3.4 The retention-time window used to make identifications should be based upon
measurements of actual retention-time variations of standards over the course of a day.
Three times the standard deviation of a retention time for a compound can be used to
calculate a suggested window size; however, the experience of the analyst should weigh
heavily in the interpretation of chroinatograms.
10.3.5 If the response forthe peak exceeds the working range of the system, dilute the sample
with mobile phase and reanalyze.
10.3.6 If the measurement of the peak response is prevented by the presence of interferences,
cleanup is required.
11. ÔALCULAUONS
11.1 Determine the concentration of analytes in the sample.
11.1.1 Calculate the amount of analytes injected from the peak response using the calibration
curve or calibration factor in Section 8.2.2. The concentration in the sample can be
calculated from the equation:
EquatIon 3
( AXV, )
Co .centratioas, pgjL , ,
wharn
A - Aaow.t of analyses btjected in nanogranis.
- Foliate of extract injected in pg/L
V 1 - Valuate of tcwi extract b, ig/L
Vs - VobmseofremacterlinmL
12. METHOD PERFORMANCE
12.1 The EDLs and associated chromatographic conditions for the analytes are listed in Table 1. The
EDL is defined as the minim m response being equal to 5 times the background noise, assuming
a 2-mL final extract volume of a 1-L sample and an HPLC injection volume of 50 pL.
12.2 Single-operator accuracy and precision studies were conducted by Environmental Science and
Engineering, Inc.,’ in the designated matrices. The results of these studies are presented in
Table 2.
566
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Method 642
References
1. ASTM Annual Book of Standards, Part 31, D3694, “Standard Practice for Preparation of Sample
Containers and for Preservation,” American Society for Testing and Materials, Philadelphia,
Pennsylvania, p. 679, 1980.
2. “Carcinogens - Working with Carcinogens,” Department of Health, Education, and Welfare,
Public Health Service, Center for Disease Control, National Institute for Occupational Safety and
Health, Publication No. 77-206, Aug. 1977.
3. “OSHA Safety and Health Standards, General Industry” (29 CFR 1910), Occupational Safety and
Health Administration, OSHA 2206 (Revised, January 1976).
4. “Safety in Academic Chemistry Laboratories,” American Chemical Society Publication,
Committee on Chemical Safety, 3rd Edition, 1979.
5. ASTM Annual Book of Standards, Part 31, D3370, “Standard Practice for Sampling Water,”
American Society for Testing and Materials, Philadelphia, PA, p. 76, 1980.
6. Test procedures for Pesticides in Wastewaters, EPA Contract Report No. 68-03-2897.
Unpublished report available from U.S. Environmental Protection Agency, Environmental
Monitoring and Support Laboratory, Cincinnati, Ohio.
7. “Handbook for Analytical Quality Control in Water and Wastewater Laboratories,”
EPA-600/4-79-019, U.S. Environmental Protection Agency, Environmental Monitoring and
Support Laboratory - Cincinnati, Ohio, March 1979.
8. Beernaert, H., Determination of Biphenyl and Ortho-Phenyiphenol in Citrus Fruits by Gas
Chromatography, Journal of Chromatography, 77: 331-8, 1973.
9. “Evaluation of Ten Pesticide Methods” U.S. Environmental Protection Agency, Contract No.
68-03-1760, Task No. 11, U.S. Environmental Monitoring and Support Laboratory, Cincinnati,
Ohio.
567
-------
Method 642
Table 1. Chromatographic Conditions and Estimated Detection Limits
Estimated
Retention rime (mi i i) Detection
Limit
Parameter Column 1 Column 2 (pg/Li
o-Theuyli henol 7.7 11.3 .01
Biphenyl 18.8 165 .04
Column 1: Reverse-phase cohunn, 4.6mm ID by 250 mm long; 10 i, Perkin-Elmer IIC-ODS Sil-X or
equivalent; isocratic elution for 5 minutes using 40% acetonitrile in water, then linear gradient elution
to 100% acetonitrile over 25 minutes; flow rate of 0.5 mL/min.
Column 2: Reverse-phase column, 4.6 mm ID by 250 mm long; 6 to 7 i, Dupont Zorbax ODS or
equivalent; Isocratic elution for 3 minutes using 50% methanol in water, then linear gradient to 80-percent
methai l over 10 mimj ; flow rate 1.0 mL/min.
Table 2. Single-Operator Accuracy and Precision
Sp& Average Standard
Rang. Number of Peicent Deviation
Parameter (pg/Li Repicates R.covery** (%)
o-Pbeny lphenol 2.5 7 102.3 36.3
6,500 7 94.1 6.3
Biphenyl 2.4 7 86.3 16.2
6,300 7 100.7 9.9
* POTW effluent was used in this study.
No cleanup was employed in validation studies.
568
-------
Method 642
Retention Time (minutes)
20.0
Figure 1. Liquid Chromatogram of Wastewater Extract Fortified With
o-PhenylpheflOl and Biphenyl (Column 1).
A52 9
/
B phenyl
0 2.0 4.0 6.0 8.0
10.0
12.0
14.0
18.0
569
-------
Method 643
The Determination of
Ben tazon in Municipal and
Industrial Waste water
-------
Method 643
The Determination of Ben tazon in Municipal and Industrial
Waste water
1. SCOPE AND APPLICATiON
1.1 This method covers the determination of bentazon in municipal and industrial wastewater.
Parameter CAS No.
Bentazon (Basagran) 25057-89-0
1.2 The estimated detection limit (EDL) for bentazon is listed in Table 1. The EDL was calculated
from the minimum detectable response being equal to 5 times the background noise using a 5-mL
final extract volume of a 1-L sample and an injection volume of 100 jiL. The EDL for a specific
wastewater may be different depending on the nature of interferences in the sample matrix.
1.3 This is a high performance liquid chromatographic (HPLC) method applicable to the determination
of bentazon in municipal and industrial discharges. When this method is used to analyze
unfamiliar samples for bentazon, compound identification should be supported by at least one
additional qualitative technique. This method describes analytical conditions for a second HPLC
column that can be used to confirm measurements made with the primary column.
1.4 This method is restricted to use by or under the supervision of analysts experienced in the
operation of liquid chromatographs and in the interpretation of liquid chromatograms.
2. SUMMARY OF METHOD
2.1 Bentazon is removed from an acidified sample matrix by extraction with methylene chloride. The
extract is discarded after back extraction with aqueous base. HPLC conditions are described
which permit the separation and measurement of bentazon in the aqueous extract.
3. INTERFERENCES
3.1 Solvent, reagents, glassware, and other sample-processing hardware may yield discrete artifacts
and/or elevated baselines causing misinterpretation of liquid chromatograms. All of these
materials must be demonstrated to be free from interferences under the conditions of the analysis
by running laboratory reagent blanks as described in Section 9.1
3.1.1 The use of high-purity reagents and solvents helps to minimize interference problems.
Purification of solvents by distillation in all-glass systems may be required.
3.1.2 Glassware must be scrupulously cleaned.’ Clean all glassware as soon as possible after
use by rinsing with the last solvent used in it. This should be followed by detergent
washing with hot water and rinses with tap water and reagent water. It should then be
drained dry and heated in a muffle furnace at 400°C for 15 to 30 minutes. Solvent rinses
with acetone and pesticide-quality hexane may be substituted for the heating. Volumetric
573
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Method 643
ware should not be heated in a muffle furnace. After drying and cooling, glassware
should be sealed and stored in a clean environment to prevent any accumulation of dust
or other contaminants. Store the glassware inverted or capped with aluminum foil.
3.2 Matrix interferences may be caused by UV-active contaminants that are coextracted from the
samples. The extent of matrix interferences will vary considerably from source to source,
depending upon the nature and diversity of the industrial complex or municipality being sampled.
Unique samples may require additional cleanup approaches to achieve the detection limit listed in
Table 1.
4. SAFETY
4.1 The toxicity or carcinogenicity of each reagent used in this method has not been precisely defined;
however, each chemical compound should be treated as a potential health hazard. From this
viewpoint exposure to these chemicals must be reduced to the lowest possible level by whatever
means available. The laboratory is responsible fur maintaining a current awareness file of OSHA
regulations regarding the safe handling of the chemicals specified in this method. A reference file
of material data handling sheets should also be made available to all personnel involved in the
chemical analysis. Additional references to laboratory safety are available and have been
identified ’ for the information of the analyst.
5. APPARATUS AND EQUIPMENT
5.1 Sample containers: Narrow-mouth glass bottles, 1-L or 1-quart volume, equipped with
polytetrafluoroethylene (FWE)-lined screw-caps. Wide-mouth glass bottles, 1-quart volume,
equipped with Ffl E-Iined screw-caps may also be used. Prior to use, wash bottles and cap liners
with detergent and rinse with tap and reagent water. Allow the bottles and cap liners to air dry,
then muffle the bottles at 400°C for 1 hour. After cooling, rinse the bottles and cap liners with
hexane, seal the bottles, and store in a dust-free environment.
5.1.1 Automatic sampler (optional): Must incorporate glass sample containers for the
• collection of a minimum of 250 mL. Sample containers must be kept refrigerated at 4°C
and protected from light during compositing. If the sampler uses a peristaltic pump, a
minimum length of compressible silicone rubber tubing may be used. Before use,
however, the compressible tubing should be thoroughly rinsed with methanol, followed
by repeated rinsings with reagent water to minimize the potential for contamination of
the sample. An integrating flow meter is required to collect flow-proportional
composites.
5.2 141gb performance liquid chromatography (HPLC) apparatus: Analytical system complete with
liquid chromatograph and all required accessories including syringes, analytical columna, and
mthile phases. The system must be compatible with the specified detectors and strip-chart
recorder. A data system is recommended for measuring peak areas.
5.2.1 Gradient pumping system, constant flow.
5.2.2 Injector valve (Itheodyne 7125 or equivalent) with 100-pL loop.
5.2.3 Column 1: 250 mm long by 4.6 mm ID, stainless steel, packed with reverse-phase
Ultrasphere O1)S, 10 i, or equivalent.
574
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Method 643
5.2.4 Column .2: 300 mm long by 4.0 mm ID, packed with reverse phase ji Bondapak C18,
(Waters Associates), or equivalent.
5.2.5 Ultraviolet detector, variable wavelength, capable of monitoring at 344) nm.
5.2.6 Strip-chart recorder compatible with detector, 250 mm. (A data system for measuring
peak areas is recommended.)
5.3 Miscellaneous.
5.3.1 Balance: analytical, capable of accurately weighing to the nearest 0.0001 g.
5.3.2 Separatory funnels: 2-L, and 250-mL, equipped with PTFE stopcocks.
5.3.3 Standard solution storage containers: 15-mL bottles with PTFE-lined screw-caps.
5.3.4 Pasteur pipettes with bulbs.
6. REAGENTS AND CONSUMABLE MA TERIALS
6.1 Reagents.
6.1.1 Acetone, hexane, methanol, and methylene chloride: Demonstrated to be free of analytes
and interferences.
6.1.2 Reagent water: Reagent water is defined as a water in which an interferent is not
observed at the method detection limit of each parameter of interest.
6.1.3 Sodium hydroxide solution (0. iN): Dissolve 0.4 g of NaOH in reagent water and dilute
to 100 mL.
6.1.4 Sodium chloride: ACS, crystals.
6.1.5 Sodium thiosulfate: ACS, granular.
6.1.6 Sulfuric acid solution (1 ÷ 1): Slowly add 50 mL of H 2 S0 4 (specific gravity 1.84) to
50 mL of reagent water.
6.1.7 Sodium hydroxide solution (6N): Dissolve 24 g of NaOH in reagent water and dilute to
100 mL.
6.1.8 Acetate buffer solution: Dissolve 0.41 g of anhydrous sodium acetate (ACS) and 1.5 mL
of glacial acetic acid (ACS) in 100 niL of reagent water.
6.1.9 Glacial acetic acid: ACS.
6.1.10 HPLC mobile phase buffer (pH 4.7, 0.062 M acetate): Dissolve 0.87 g of anhydrous
sodium acetate (ACS) and 3.0 mL of glacial acetic acid (ACS) in 1 L of reagent water.
6.2 Standard stock solution (1.00 gI L): This solution may be purchased as a certified solution or
prepared from a pure standard material using the following procedures.
6.2.1 Prepare the stock standard solution by accurately weighing about 0.0100 g of pure
material. Dissolve the material in pesticide-quality methanol, dilute to volume in a
10-mL volumetric flask. Larger volumes can be used at the convenience of the analyst.
When compound purity is certified at 96% or greater, the weight can be used without
correction to calculate the concentration of the stock standard. Commercially prepared
stock standards can be used at any concentration if they are certified by the manufacturer
or by an independent source.
575
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Method 643
6.2.2 Transfer the stock standards to a Fl’FE-sealed screw-cap bottles. Store at 4°C and
protect from light. Stock standards should be checked frequently for signs of degradation
or evaporation, especially just prior to preparing calibration standards from them.
6.2.3 Stock standards must be replaced after 6 months, or when comparison with quality
control check samples indicates a problem.
7. SAMPLE COLLECTiON, PRESERVATiON, AND STORAGE
7.1 Collect all samples in duplicate. Grab samples must be collected in glass containers.
Conventional sampling practic& should be followed, except that the bottle must not be prewashed
with sample before collection.
7.2 The samples must be iced or refrigerated at 4°C from the time of collection until extraction. If
the samples will not be extracted within 48 hours of collection, the sample should be adjusted to
a pH range of 6.0 to 8.0 with sodium hydroxide or sulfuric acid and add 35 mg of sodium
thlosulfate per liter of sample for each part per million of free chlorine.
7.3 All skn $es must be extracted within 7 days and completely analyzed within 30 days of
extraction.’
8. CALIBRATION
8.1 Establish liquid chromatographic operating parameters equivalent to those indicated in Table 1.
8.2 Prepare calibration standards at a minimum of three concentration levels of bentazon by
adding volumes of the stock standard to a volumetric flask and diluting to volume with HPLC
mobile pluse (35% methanol in HPLC mobile phase buffer or 40% methanol in HPLC mobile
phase buffer). One of the standards should be at a concentration near, but greater than, the EDL,
and the other concentrations should correspond to the expected range of concentrations found in
real san les or should define the working range of the detector.
8.3 Using injections of 100 1 iL of each calibration standard, tabulate peak height or area response
against the injected. The results are used to prepare a calibration curve for the analytes.
Alternatively, if the ratio of r onse to amount injected (calibration factor) is a constant over the
working range (<10% relative st Iard deviation, RSD), linearity of the calibration curve can
beassuniedandtheaverage ratioorcallbrationfactor canbeused inpiaceofa calibrationcurve.
8.4 The working calibration curve or calibration factor must be verified on each working day by the
_________of one or more calibration standards. If the response for bentazon varies from the
predicted response by more than ± 10%, the test must be repeated using a fresh calibration
standard. Alternatively, a new calibration curve or factor must be prepared.
8.5 Before using any deanup procedure, the analyst must process a series of calibration standards
through the procedure to validate elution patterns and the absence of interferences from the
576
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Method 643
9. QUALITY CONTROL
9.1 Monitoring for interferences.
9.1.1 Analyze a laboratory reagent blank each time a set of samples is extracted. A laboratory
reagent blank is an aliquot of reagent water. If the reagent blank contains a reportable
level of bentazon, immediately check the entire analytical system to locate and correct
for possible interferences and repeat the test.
9.2 Assessing accuracy.
9.2.1 After every ten samples, and preferably in the middle of each day, analyze a laboratory
control standard. Calibration standards may not be used for accuracy assessments and
the laboratory control standard may not be used for calibration of the analytical system.
9.2.1.1 Laboratory control standard concentrate: From stock standards prepared as
described in Section 6.2, prepare a laboratory control standard concentrate
that contains bentazon at a concentration of 2 ig/ L in methanol.
9.2.1 .2 Laboratory control standard: Using a pipette or microliter syringe, add 50.0
iL of the laboratory control standard concentrate to a 1-L aliquot of reagent
water. 7
9.2.1.3 Analyze the laboratory control standard as described in Section 10. Calculate
the percent recovery (P. 1 ) with the equation:
Equation 1
1005
I
where
5, = The analytical rerults from the laboratory control standard, in g/L
= The kno i concentration of the spike, in &g/L
9.2.2 At least annually, the laboratory should participate in formal performance evaluation
studies, where solutions of unknown concentrations are analyzed and the performance of
all participants is compared. 7
9.3 Assessing precision.
9 3 1 Precision assessments for this method are based upon the analysis of field duplicates
(Section 7.1). Analyze both samples for at least 10% of all samples. To the extent
practical, the samples for duplication should contain reportable levels of bentazon.
9.3.2 Calculate the relative range 7 (RRJ with the equation:
577
-------
Method 643
Equation 2
100R 1
RR = _____
I
where
= The absolute difference between the displicate nieasureinen&c X and 12, in g/L
= The average concentration found ( ), in g/L
9.3.3 Individual relative range measurements are pooled to determine average relative range
or to develop an expression of relative range as a function of concentration.
10. PROCEDURE
10.1 Sample extraction.
10.1.1 Mark the water meniscus on the side of the sample bottle for later determination of
sample volume. Pour the entire sample into a 2-L separatory funnel. Check the pH of
the sample with wide-range pH paper and adjust to within the range of 2.5 to 3.5 with
sulfuric acid. Add 200 g of sodium chloride and mix to dissolve.
10.1.2 Add 60 niL of methylene chloride to the sample bottle and shake for 30 seconds to rinse
the walls. Transfer the solvent to the separatory funnel and extract the sample by shaking
the funnel for 2 minutes with periodic venting to release vapor pressure. Allow the
organic layer to separate from the water phase for a minimum of 10 minutes. If the
emulsion interf ce between layers is more than one-third the volume of the solvent layer,
the analyst must employ mechanical techniques to complete the phase separation. The
optimum technique depends on the sample, but may include stirring, filtration of the
emulsion through glass wool, or centrifugation. Collect the extract in a 250-mL
separatory funnel.
10.1.3 Add an additional 60-mL volume of methylene chloride to the sample bottle and complete
the extraction procedure a second time, combining the extracts in the 250-mL separatory
funnel.
10.1.4 Perform a third extraction in the same manner. Add 2 mL of 0.1M NaOH in reagent
water to the 250-mL separatory funnel, and extract by shaking the funnel for 2 minutes
with periodic venting to release vapor pressure. Allow the organic layer to separate from
the water phase for a minimum of 10 minutes. Drain the methylene chloride into a
250-mL Erlenmeyer flask. Transfer the aqueous layer with a Pasteur pipette to a 5-mL
volumetric flask.
10.1.5 Add the methylene chloride back to the 250-mL separatory funnel, and extract with an
additional 2 mL of 0. IM NaOH. Combine the extracts in the 5-mL volumetric flask.
10.1.6 Add two drops of glacial acetic acid to the volumetric flask, and dilute to volume with
acetate buffer solution (Section 6.1.7).
578
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Method 643
10.1.7 Determine the original sample volume by refilling the sample bottle to the mark and
transferring the liquid to a 1,000-mi. graduated cylinder. Record the sample volume to
the nearest 5 mL.
10.2 Cleanup and separation.
10.2.1 The cleanup procedure recommended in this method involves the back extraction of a
methylene chloride extract with aqueous base, and has been used for the analysis of
various clean waters and industrial effluents. If additional cleanup is required, a 1-L
sample is adjusted to pH 12 with 6N NaOH and extracted with three 60-mL aliquots of
methylene chloride in a 2-L separatory funnel. The methylene chloride extracts are
discarded and the aqueous sample adjusted to pH range of 2.5 to 3.5 with 1:1 sulfuric
acid solution for re-extraction as in Section 10.1.1. If additional cleanup is required, or
if particular circumstances demand the use of an alternative cleanup procedure, the
analyst must determine the elution proffle and demonstrate that the recovery for each
compound of interest is no less than 85%.
10.3 Liquid chromatography analysis.
10.3.1 Table 1 summarizes the recommended operating conditions for the liquid chromatograph.
Included in this table are the estimated retention times and estimated detection limit that
can be achieved by this method. An example of the separation achieved by Column 2
is shown in Figure 1. Other columns, chromatographic conditions, or detectors may be
used if data quality comparable to Table 2 is achieved.
10.3.2 Calibrate the system daily as described in Section 8.
10.3.3 Inject 100 iL of the sample extract. Monitor the column eluent at 340 nm. Record the
resulting peak size in area or peak height units.
10.3.4 The retention-time window used to make identifications should be based upon
measurements of actual retention-time variations of standards over the course of a day.
Three times the standard deviation of a retention time for a compound can be used to
calculate a suggested window size; however, the experience of the analyst should weigh
heavily in the interpretation of chromatograms.
10.3.5 If the response for the peak exceeds the working range of the system, dilute the sample
with mobile phase and reanalyze.
10.3.6 If the measurement of the peak response is prevented by the presence of interferences,
additional cleanup is required.
11. CALCULA T1ONS
11.1 Determine the concentration of bentazon in the sample.
11.1.1 Calculate the amount of bentazon injected from the peak response using the calibration
curve. The concentration in the sample can be calculated from the equation:
579
-------
Method 643
EquatIon 3
( A)(V )
Concentration, pg/L - _____
(V,)(V,)
where
A = Amowit of material injecte4, in ng
V 1 - Volume of emuct iitjected in pL
Vt - Volume of total e. ract, in pL
V 1 Volume of water exiractei4 in ,nL
11.2 Report results in micrograms per liter without correction for recovery data. When duplicate and
spiked samples are analyzed, report all data obtained with the sample results.
12. METHOD PERFORMANCE
12.1 The EDL and associated chromatographic conditions for bentazon are listed in Table i. The
EDL is defined as the minimum response being equal to 5 times the background noise, assuming
a 5-mL final extract volume of a 1-L sample and an HPLC injection volume of 100 L.
12.2 Single-operator accuracy and precision studies were conducted by Environmental Science and
Engineering, Inc., 6 in the designated matrices. The results of these studies are presented in
Table 2.
580
-------
Method 643
References
1. ASTM Annual Book of Standards, Vol. 11.02, D3694, “Standard Practice for Preparation of
Sample Containers and for Preservation,” American Society for Testing and Materials,
Philadelphia, Pennsylvania, 1986.
2. “Carcinogens - Working with Carcinogens,” Department of Health, Education, and Welfare,
Public Health Service, Center for Disease Control, National Institute for Occupational Safety and
Health, Publication No. 77-206, Aug. 1977.
3. “OSHA Safety and Health Standards, General Industry” (29 CFR 1910), Occupational Safety and
Health Administration, OSHA 2206 (Revised, January 1976).
4. “Safety in Academic Chemistry Laboratories,” American Chemical Society Publication,
Committee on Chemical Safety, 3rd Edition, 1979.
5. ASTM Annual Book of Standards, Vol. 11.01, D3370, “Standard Practice for Sampling Water,”
American Society for Testing and Materials, Philadelphia, Pennsylvania, 1986.
6. Test procedures for Pesticides in Wastewaters, EPA Contract Report No. 68-03-2897 (In
Preparation). Unpublished report available from U.S. Environmental Protection Agency,
Environmental Monitoring and Support Laboratory, Cincinnati, Ohio.
7. “Handbook for Analytical Quality Control in Water and Wastewater Laboratories,”
EPA-600/4-79-019, U.S. Environmental Protection Agency, Environmental Monitoring and
Support Laboratory - Cincinnati, Ohio 45268, March 1979.
8. “Evaluation of Ten Pesticide Methods,” U.S. Environmental Protection Agency Contract No.
68-03-1760, Task No. 11, U.S. Environmenal Monitoring and Support Laboratory, Cincinnati,
Ohio.
581
-------
Method 643
Table 1. Liquid Chromatography of Bentazon
Parameter Retention Time (miii Estimated Detection Limit
Cc*imn I Column 2 (pg/Li
Bentazon 7.9 4.3 1.1
HPLC Column 1: Reverse-phase column, 250 mm long by 4.6 mm ID, stainless steel, packed with 10
i Ultrasphere ODS or equivalent. Isocratic elution with 35% methanol/65% buffer; flow rate 2.0
nillmm.
HPLC Column 2: Reverse-phase column, 300 mm long by 4 mm ID, stainless steel, packed with a
Bondapak C18, 10 u, Waters Associates or equivalent. Linear gradient elution of 40% methanol/60%
buffer to 52% methanol/48% buffer over 9 minutes; flow rate 1 inL/min.
Table 2. SIngle-Laboratory Accuracy and Precision
Parameter Matrix Range No. of Average % Standard
Type pg t Repicates Recovery Deviation (%i
Benwon 1 125 7 85.1 4.8
2 20,400 7 88.4 8.4
1= 50% industrial effluent +50% POTW effluent
2 = 100% industrial effluent
582
-------
Method 643
Figure 1. HPLC of Bentazon (Column 2).
Bentazon
/
0 1.0 2.0 3.0 4.0 5.0 6.0
Retention Time (minutes)
A .O -7O
583
-------
Method 644
The Determination of
Picloram in Municipal and
industrial Waste water
-------
Method 644
The Determination of Picloram in Municipal and Industrial
Waste water
SCOPE AND APPLICA T1ON
1.1 This method covers the determination of picloram in municipal and industrial wastewater.
Parameter CAS No.
Picloram 1918-02-1
1.2 The estimated detection limit (EDL) for picloram is listed in Table 1. The EDL was calculated
from the minimum detectable response being equal to 5 times the background noise using a
100- &L injection. The EDL for a specific wastewater may be different depending on the nature
of interferences in the sample matrix.
1.3 This is a high performance liquid chromatographic (HPLC) method applicable to the determination
of picloram in municipal and industrial discharges. When this method is used to analyze
unfamiliar samples for picloram, compound identification should be supported by at least one
additional qualitative technique. -
1.4 This method is restricted to use by or under the supervision of analysts experienced in the
operation of liquid chromatographs and in the interpretation of liquid chromatograms.
2. SUMMARY OF METHOD
2.1 Picloram is removed from the acidified sample matrix by extraction with methylene chloride. The
extract is dried, exchanged to HPLC mobile phase, and analyzed by HPLC with ultraviolet (UV)
detection. An alkaline back-extraction is used as necessary to eliminate interferences which may
be encountered.
3. INTERFERENCES
3.1 Solvent, reagents, glassware, and other sample-processing hardware may yield discrete artifacts
and/or elevated baselines causing misinterpretation of liquid chromatograms. All of these
materials must be demonstrated to be free from interferences under the conditions of the analysis
by running laboratory reagent blanks as described in Section 9.1.
3.1.1 The use of high-purity reagents and solvents helps to minimize interference problems.
Purification of solvents by distillation in all-glass systems may be required.
3.1.2 Glassware must be scrupulously cleaned. 1 Clean all glassware as soon as possible after
use by rinsing with the last solvent used in it. This should be followed by detergent
washing with hot water and rinses with tap water and reagent water. It should then be
drained dry and heated in a muffle furnace at 400°C for 15 to 30 minutes. Solvent rinses
with acetone and pesticide-quality hexane may be substituted for the heating. Volumetric
ware should not be heated in a muffle furnace. After drying and cooling, glassware
587
-------
Method 644
should be sealed and stored in a clean environment to prevent any accumulation of dust
or other contaminants. Store the glassware inverted or capped with aluminum foil.
3.2 Matrix interferences may be caused by UV-active contaminants that coelute with picloram. The
extent of matrix interferences will vary considerably from source to source, depending upon the
nature and diversity of the industrial complex or municipality being sampled. While general
cleanup procedures are provided as part of this method, unique samples may require additional
cleanup approaches to achieve the detection limit listed in Table 1.
4. SAFETY
4.1 The toxicity or carcinogenicity of each reagent used in this method has not been precisely defined;
however, each chemical compound should be treated as a potential health hazard. From this
viewpoint, exposure to these chemicals must be reduced to the lowest possible level by whatever
means available. The laboratory is responsible for maintaining a current awareness file of OSHA
regulations regarding the safe handling of the chemicals specified in this method. A reference file
of material data handling sheets should also be made available to all personnel involved in the
chemical analysis. Additional references to laboratory safety are available and have been
identified for the information of the analyst.
5. APPARA TUS AND EQwPMENT
5.1 Sample containers: Narrow-mouth glass bottles, 1-L or 1-quart volume, equipped with
polytetrafluoroethylene (PTFE)-Iined screw-caps. Wide-mouth glass bottles, 1-quart volume,
equipped with PTFE-lined screw-caps may also be used. Prior to use, wash bottles and cap liners
with detergent and rinse with tap and reagent water. Allow the bottles and cap liners to air dry,
then muffle at 400°C for 1 hour. After cooling, rinse the bottles and cap liners with hexane, seal
the bottles, and store in a dust-free environment.
5.1.1 Automatic sampler (optional): Must incorporate glass sample containers for the
collection of a minimum of 250 mL. Sample containers must be kept refrigerated at 4°C
and protected from light during compositing. If the sampler uses a peristaltic pump, a
minimum length of compressible silicone rubber tubing may be used. Before use,
however, the compressible tubing should be thoroughly rinsed with methanol, followed
by repeated rinsings with reagent water to minimize the potential for contamination of
the sample. An integrating flow meter is required to collect flow-proportional
composites.
5.2 Kuderna-Danish (K-D) glassware.
5.2.1 Synder column: Three-ball macro (Kontes K-503000-0121 or equivalent) and two-ball
micro (Kontes K-569001-0219 or equivalent).
5.2.2 Concentrator tube: 10-mL, graduated (Kontes K-570050-l025 or equivalent) with
ground-glass stopper.
5.2.3 Evaporative flask: 500-mL (Kontes K-570001-0500 or equivalent). Attach to
concentrator tube with springs.
5.3 High performance liquid chromatography (HPLC) apparatus: Analytical system complete with
liquid chromatograph and all required accessories including syringes, analytical columns, and
588
-------
Method 644
mobile phases. The system must be compatible with the specified detector and strip-chart
recorder. A data system is recommended for measuring peak areas.
5.3.1 Isocratic pumping system, constant flow.
5.3.2 Injector valve (Rheodyne 7125 or equivalent) with 100-FL loop.
5.3.3 Column: 250 mm long by 4.6 mm ID, stainless steel, packed with reverse-phase
Ultrasphere ODS, 10
5.3.4 Ultraviolet detector, variable wavelength, capable of monitoring at 225 nm.
5.3.5 Strip-chart recorder compatible with detector, 250 mm. (A data system for measuring
peak areas is recommended.)
5.4 Chromatographic column: 300 mm long by 10 mm ID Chromaflex, equipped with coarse-fitted
bottom plate and PTFE stopcock. (Kontes K-420540-0213 or equivalent).
5.5 Drying column: Approximately 400 mm long by 20 mm ID borosilicate glass, equipped with
coarse-fitted bottom plate.
5.6 Miscellaneous.
5.6.1 Balance: Analytical, capable of accurately weighing to the nearest 0.0001 g.
5.6.2 Separatory funnels: 2-L, 500-mL, and 250-mL, equipped with PTFE stopcocks.
5.6.3 Boiling chips: Approximately 10140 mesh. Heat to 400°C for 30 minutes or perform
a Soxhiet extraction with methylene chloride for 2 hours.
5.6.4 Water bath: Heated with concentric ring cover, capable of temperature control (±2°C).
The bath should be used in a hood.
5.6.5 Pasteur pipettes and bulbs.
5.6.6 Standard solution storage containers: 15-mL bottles with PTFE-lined screw-caps.
6. REA GENTS AND CONSUMABLE MA TERIALS
6.1 Reagents.
6.1.1 Acetone, hexane, methanol, and methylene chloride: Demonstrated to be free of analytes
and interferences.
6.1.2 Reagent water: Reagent water is defined as a water in which an interferent is not
observed at the method detection limit of each parameter of interest.
6.1.3 Sodium hydroxide (NaOH) solution (0.3N): Dissolve 12 g NaOH in reagent water and
dilute to l000mL.
6.1.4 Sodium sulfate: Granular, anhydrous. Condition by heating at 400°C for 4 hours in a
shallow tray.
6.1.5 Sodium chloride: ACS, crystals.
6.1.6 Sulfuric acid (H 2 S0 4 ) solution (1+1): Add a measured volume of concentrated H 2 S0 4
to an equal volume of reagent water.
6.1.7 HPLC buffer (pH 2, 0.1M phosphate): Dissolve 5.83 g of KH 2 PO 4 (ACS) and 3.9 mL
of 85% phosphoric acid (ACS) in 1 L of reagent water.
589
-------
Method 644
6.1.8 HPLC mobile phase: Add 570 mL of HPLC buffer solution to a 1-1. volumetric flask
and dilute to volume with methanol.
6.2 Standard stock solutions (1.00 pg/ zL): These solutions may be purchased as a certified solution
or prepared from the pure standard material using the following procedures.
6.2.1 Prepare the stock standard solution by accurately weighing about 0.0100 g of pure
material. Dissolve the material in pesticide-quality methanol and dilute to volume in a
10-mL volumetric flask. Larger volumes can be used at the convenience of the analyst.
When compound purity is certified at 96% or greater, the weight can be used without
correction to calculate the concentration of the stock standard. Commercially prepared
stock standards can be used at any concentration if they are certified by the manufacturer
or by an independent source.
6.2.2 Transfer the stock standard to a PTFE-sealed screw-cap bottle. Store at 4°C and protect
from light. The stock standard should be checked frequently for signs of degradation or
evaporation, especially just prior to preparing calibration standards from it.
6.2.3 The stock standard must be replaced after 6 months, or when comparison with a quality
control check sample indicates a problem.
7. SAMPLE COLLECTION, PRESERVATION, AND STORAGE
7.1 Collect all samples in duplicate. Grab samples must be collected in glass containers.
Conventional sampling practic& should be followed, except that the bottle must not be prewashed
with sample before collection.
7.2 The samples must be iced or refrigerated at 4°C from the time of collection until analysis.
Chemical preservatives should not be used in the field unless more than 24 hours will elapse
before delivery to the laboratory. if the samples will not be analyzed within 48 hours of
collection, the sample should be adjusted to a pH range of 1.0 to 3.0 with sodium hydroxide or
sulfuric acid.
7.3 All samples must be extracted within 7 days of collection and analyzed within 30 days of
extraction.’
8. CALiBRATiON
8.1 Establish liquid chromatographic operating param ers equivalent to those indicated in Table 1.
82 Prepare calibration standards at a minimum of three concentration levels of picloram by adding
volumes of the stock standard to a volumetric flask and diluting to volume with HPLC mobile
phase. One of the standards should be at a concentration near, but greater than, the EDL, and
the other concentrations should correspond to the expected range of concentrations found in real
samples or should define the working range of the detector.
8.3 Using injections of 100 pL of each calibration standard, tabulate peak height or area response
against the mass injected. The results are used to prepare a calibration curve for picloram.
Alternatively, if the ratio of response to amount injected (calibration factor) is a constant over the
working range (<10% relative standard deviation), linearity of the calibration curve can be
assumed and the average ratio or calibration factor can be used in place of a calibration curve.
590
-------
Method 644
8.4 The working calibration curve or calibration factor must be verified on each working day by the
measurement of one or more calibration standards. If the response for picloram varies from the
predicted response by ±10%, the test must be repeated using a fresh calibration standard.
Alternatively, a new calibration curve or factor must be prepared.
9. QuAUTY CONTROL
9.1 Monitoring for interferences.
9.1.1 Analyze a laboratory reagent blank each time a set of samples is extracted. A laboratory
reagent blank is an aliquot of reagent water. If the reagent blank contains a reportable
level of picloram, immediately check the entire analytical system to locate and correct
for possible interferences and repeat the test.
9.2 Assessing accuracy.
9.2.1 After every ten samples, and preferably in the middle of each day, analyze a laboratory
control standard. Calibration standards may not be used for accuracy assessments and
the laboratory control standard may not be used for calibration of the analytical system.
9.2.1.1 Laboratory control standard concentrate: From the stock standard prepared
as described in Section 6.3, prepare a laboratory control standard concentrate
that contains picloram at a concentration of 2 j&gJmL in methanol or other
suitable solvent. 7
9.2.1.2 Laboratory control standard: Using a pipette add 1.0 mL of the laboratory
control standard concentrate to a 1-L aliquot of reagent water.
9.2.1.3 Analyze the laboratory control standard as described in Section 10. Calculate
the percent recovery (P 1 ) with the equation:
Equation 1
P=
loos
‘
T 1
where
S 1 The
analytical
results from the
laboratory
control
standard,
in
pg/L
The
known co
ncentration of the
spike, in
ig/L
9.2.2 At least annually, the laboratory should participate in formal performance evaluation
studies, where solutions of unknown concentrations are analyzed and the performance of
all participants is compared. 7
9.3 Assessing precision.
9.3.1 Precision assessments for this method are based upon the analysis of field duplicates
(Section 7.1). Analyze both sample bottles for at least 10% of all samples. To the extent
practical, the samples for duplication should contain reportable levels of picloram.
9.3.2 Calculate the relative range 7 (RR 1 ) with the equation:
591
-------
Method 644
Equation 2
100R ,
where
R = the akobae thfference between the duplicate measurements ’ X 1 and X 2 , in ig/L
( x+x
the ave ge concentration finin i 1 2 J in pg/L
9.3.3 Individual relative range measurements are pooled to determine average relative range
or to develop an expression of relative range as a function of concentration.
10. PROCEDURE
10.1 Sample extraction.
10.1.1 Mark the water meniscus on the side of the sample bottle for later determination of
sample volume. Pour the entire sample into a 2-L separatory funnel. Check the pH of
the sample with wide-range pH paper and adjust to within the range of 1.5 to 2.5 with
sulfuric acid. Add 200 g of sodium chloride and mix to dissolve.
10.1.2 Add 60 mL of methylene chloride to the sample bottle and shake for 30 seconds to rinse
the walls. Transfer the solvent to the separatory funnel and extract the sample by shaking
the funnel fur 2 minutes with periodic venting to release vapor pressure. Allow the
organic layer to separate from the water phase for a minimum of 10 minutes. If the
emulsion intethce between layers is more than one-third the volume of the solvent layer,
the analyst must employ mechanical techniques to complete the phase separation. The
optimum technique depends on the sample, but may include stirring, filtration of the
emulsion through glass wool, or centrifugation. Collect the extract in a 250-mL
Erlenmeyer flask.
10.1.3 Add an additional 60-mL volume of methylene chloride to the sample bottle and complete
the extraction procedure a second time, combining the extracts in the Erlenmeyer flask.
10.1.4 Perform a third extraction in the same manner. Pour the combined extract through a
drying column containing about 10 cm of anhydrous sodium sulfate. if the extract
requires cleanup, collect the extract in a 500-mL separatory funnel and proceed to
Section 10.2 (cleanup and separation). If the extract does not require cleanup, collect the
extract in a 500-mL K-D flask equipped with a 10-mL concentrator tube and proceed
with Sections 10.1.5 and 10.1.6.
10.1.5 Add one or two clean boiling chips to the flask and attach a three-ball Snyder column.
Prewet the Snyder column by adding about 1 mL of methylene chloride to the top. Place
the IC-D apparatus on a hot water bath (80 to 85°C) so that the concentrator tube is
partially immersed in the hot water and the entire lower rounded surface of the flask is
bathed in steam. Adjust the vertical position of the apparatus and the water temperature
as required to complete the concentration in 15 to 20 minutes. At the proper rate of
distillation, the balls of the column will actively chatter but the chambers will not flood.
When the apparent volume of liquid reaches 5 mL, remove the K-I) apparatus and allow
592
-------
Method 644
it to drain and cool for at least 10 minutes. Add 50 mL of methanol and a clean boiling
chip to the flask and repeat the concentration as described above. When the apparent
volume of the liquid reaches 1 mL, remove the K-D apparatus and allow it to drain and
cool for at least 10 minutes. Remove the Snyder column and rinse the flask and its lower
joint into the concentrator tube with 1 to 2 mL of methanol. A 5-niL syringe is
recommended for this operation.
10.1.6 Add a clean boiling chip to the concentrator tube. Attach a two-ball micro-Snyder
column. Prewet the micro-Snyder column by adding about 0.5 mL of methanol to the
top. Place the micro K-D apparatus on a hot water bath (80 to 85°C) so that the
concentrator tube is partially immersed in the hot water. Adjust the vertical position of
the apparatus and water temperature as required to complete the concentration in 5 to 10
minutes. At the proper rate of distillation, the balls will actively chatter but the chambers
will not flood. When the apparent volume of liquid reaches 0.5 mL, remove the K-D
apparatus and allow it to drain and cool for at least 10 minutes. Remove the
micro-Snyder column and rinse its lower joint into the concentrator tube with a small
volume of methanol. Quantitatively transfer the extract to a 25-mL volumetric flask by
means of a Pasteur pipette or other suitable device. Rinse the concentrator tube with
about 0.5 mL of methanol and add, to the volumetric flask. Adjust the final volume to
25 mL or to a volume suitable for liquid chromatography with HPLC mobile phase.
Store refrigerated if further processing will not be performed immediately. Proceed with
liquid chromatographic analysis.
10.1.7 Determine the original sample volume by refilling the sample bottle to the mark and
transferring the liquid to a 1,000-niL graduated cylinder. Record the sample volume to
the nearest 5 mL.
10.2 Cleanup and separation.
10.2.1 Cleanup procedures may not be necessary for a relatively clean sample matrix. The
cleanup procedures recommended in this method have been used for the analysis of
various clean waters and municipal effluents. If particular circumstances demand the use
of an alternative cleanup procedure, the analyst must determine the elution profile and
demonstrate that the recovery of each compound of interest is no less than that recorded
in Table 2.
10.2.2 Collect the dried extracts from Section 10.1.4 in a 500-mL separatory funnel. Add
10 mL of 0.3N NaOH and extract by shaking the funnel for 2 minutes with periodic
venting to release excess pressure. Allow a 10-minute separation time. Drain the
methylene chloride and discard. Allow 2 minutes for the aqueous layer to drain from the
walls, and collect it in a 25-niL volumetric flask.
10.2.3 Adjust the pH of the aqueous extract to 1.5 to 2.5 with sulfuric acid solution, and dilute
to volume with HPLC mobile phase.
10.2.4 Proceed with liquid chromatography as described in Section 10.3.
10.3 Liquid chromatography analysis.
10.3.1 Table I summarizes the recommended operating conditions for the liquid chromatograph.
Included in this table are the estimated retention time and estimated detection limit that
can be achieved by this method. An example of the separation achieved by this column
593
-------
Method 644
is shown in Figure 1. Figure 2 is a chromatogram of picloram in a POTW wastewater
sample. Other columns, chromatographic conditions, or detectors may be used if data
quality comparable to Table 2 is achieved.
10.3.2 Calibrate the system daily as described in Section 8.
10.3.3 Inject 100 ,LL of the sample extract. Monitor the column eluent at 225 nm. Record the
resulting peak size in area or peak height units.
10.3.4 The retention-time window used to make identifications should be based upon
measurements of actual retention-time variations of standards over the course of a day.
Three times the standard deviation of a retention time for a compound can be used to
calculate a suggested window size; however, the experience of the analyst should weigh
heavily in the interpretation of chromatograms.
10.3.5 If the response for the peak exceeds the working range of the system, dilute the sample
with mobile phase and reanalyze.
10.3.6 If therneasurement of the peak response is prevented by the presence of interferences,
further cleanup Is required.
11. CALCULA lIONS
11.1 Determinetheconcentrationofpicloraminthesample.
11.1.1 Calculate the amount of-picloram injected from the peak response using the calibration
curve or calibration factor in Section 8.2.2. The concentration in the sample can be
calculated from the equation:
Equation 3
Concentrwion, gIL =
n*nT
A = Amount of material injected, in ng
V 1 = Voiwne of extract injected, in pL
V 1 = Volume of total extract, in 1 iL
= Volume of ter extracted, in niL
11.2 Report results in micrograms per liter without correction for recovery data. When duplicate and
spiked samples are analyzed, report all data obtained with the sample results.
12. METHOD PERFORMANCE
12.1 The EDL and associated chromatographic conditions for picloram are listed in Table 1. The
EDL is defined as the minimum response being equal to 5 times the background noise, using a
100-ILL injection.
12.2 Single-operator accuracy and precision studies were conducted by Environmental Science and
Engineering, Inc.,’ in the designated matrix. The results of these studies are presented in Table 2.
594
-------
Method 644
References
1. ASTM Annual Book of Standards, Part 31, D3694, “Standard Practice for Preparation of Sample
Containers and for Preservation,” American Society for Testing and Materials, Philadelphia,
Pennsylvania, p. 679., 1980.
2. “Carcinogens - Working with Carcinogens,” Department of Health, Education, and Welfare,
Public Health Service, Center for Disease Control, National Institute for Occupational Safety and
Health, Publication No. 77-206, Aug. 1977.
3. “OSHA Safety and Health Standards, General Industry” (29 CFR 1910), Occupational Safety and
Health Administration, OSHA 2206 (Revised, January 1976).
4. “Safety in Academic Chemistry Laboratories,” American Chemical Society Publication,
Committee on Chemical Safety, 3rd Edition, 1979.
5. ASTM Annual Book of Standards, Part 31, D3370, “Standard Practice for Sampling Water,”
American Society for Testing and Materials, Philadelphia, Pennsylvania, p. 76 1980.
6. Test procedures for Pesticides in Wastewaters, EPA Contract Report è68-03-2897 Unpublished
report, available from the U.S. Environmental Protection Agency, Environmental Monitoring and
Support Laboratory, Cincinnati, Ohio.
7. “Handbook for Analytical Quality Control in Water and Wastewater Laboratories,”
EPA-600/4-79-019, U.S. Environmental Protection Agency, Environmental Monitoring and
Support Laboratory - Cincinnati, Ohio, March 1979.
8. “Evaluation of Ten Pesticide Methods,” U.S. Environmental Protection Agency, Contract No.
68-03-1760, Task No. 11, Environmental Monitoring and Support Laboratory, Cincinnati, Ohio.
595
-------
Method 644
Table 1. Liquid Chromatography of Picloram*
Retention Time Estimated Detection
Compound (mini Limit (pg/LI
Picloram 4.0 0.3
t HPLC conditions: Reverse-phase lOp Ultrasphere ODS, 4.6 mm ID by 250 mm long column; isocratic
elution; flow rate I mLlmin. Mobile Phase: 57% HPLC Buffer solution in methanol (V/V).
596
-------
Method 644
Table 2. Single-Operator Accuracy and Precision
Spike Average Standard
Matrix Range Number of Percent Deviation
Parameter Type* (pg/L) Replicates Recovery (96)
Picloram 1 25 7 93.9 9.1
1 778 7 79.0 7.7
*1 = Municipal effluent
597
-------
Method 644
/
I I I I I I
0 1.0 2.0 3.0 4.0 5.0 6.0
RMsrdIon T1m (minutes)
*52.002-71
Figure 1. HPLC of Picloram
591
-------
Mmh 644
Figure 2. HPLC of Picloram in Wastewater Extract
Picloram
0
5.0 6.0 7.0
Retention Time (minutes)
A52-O -72
599
-------
Method 645
The Determination of Certain
Amine Pesticides and Lethane
in Municipal and Industrial
Waste water
-------
Method 645
The Determination of Certain Amine Pesticides and Lethane in
Municipal and Industrial Wastewater
SCOPE AND APPLICA TION
1.1 This method covers the determination of certain amine pesticides and lethane in municipal and
industrial wastewater. The following parameters may be determined by this method.
Parameter CAS No.
Alachlor 15972-60-8
Butachlor 23184-66-9
Diphenamid 957-51-7
Fluridone 59756-60-4
Lethane 112-56-1
Norfiurazon - 27314-13-2
1.2 The estimated detection limit (EDL) for each parameter is listed in Tables 1 and 2. The EDL was
calculated from the minimum detectable response of the nitrogen/phosphorous detector equal to
5 times the gas chromatographic (GC) background noise assuming a 10 mL final extract volume
of a 1-L reagent water sample and a GC injection of 5 ,zL. The EDL for a specific wastewater
may be different depending on the nature of interferences in the sample matrix.
1.3 This is a gas chromatographic (GC) method applicable to the determination of the compounds
listed above in municipal and industrial discharges. When this method is used to analyze
unfamiliar samples for any or all of the compounds listed above, compound identifications should
be supported by at least one additional qualitative technique. Section 13 provides gas
chromatograph/mass spectrometer (GCIMS) conditions appropriate for the qualitative confirmation
of compound identifications.
1.4 This method is restricted to use by or under the supervision of analysts experienced in the
operation of gas cbromatographs and’in the interpretation of chromatograms.
2. SUMMARY OF METHOD
2.1 The amine pesticides and lethane are removed from the sample matrix by extraction with
methylene chloride. The extract is dried, exchanged into hexane, and analyzed by gas
chromatography. Column chromatography is used as necessary to eliminate interferences which
may be encountered. Measurement of the pesticides is accomplished with a nitrogen/phosphorous
specific detector.
603
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Method 645
2.2 Confirmatory analysis by gas chromatography/mass spectrometry is recommended (Section 13)
when a new or undefined sample type is being analyzed if the concentration is adequate for such
determination.
3. INTERFERENCES
3.1 Solvent, reagents, glassware, and other sample-processing hardware may yield discrete artifacts
and/or elevated baselines causing misinterpretation of gas chromatograms. All of these materials
must be demonstrated to be free from interferences under the conditions of the analysis by running
laboratory reagent blanks as described in Section 9.1
3.1.1 The use of high-purity reagents and solvents helps to minimize interference problems.
Purification of solvents by distillation in all-glass systems may be required.
3.1.2 Glassware must be scrupulously cleaned.’ Clean all glassware as soon as possible after
use by rinsing with the last solvent used in it. This should be followed by detergent
washing with hot water and rinses with tap water and reagent water. It should then be
drained dry and heated in a muffle furnace at 400°C for 15 to 30 minutes. Solvent rinses
with acetone and pesticide-quality hexane may be substituted for the muffle furnace
heating. Volumetric ware should not be heated in a muffle furnace. After drying and
cooling, glassware should be sealed and stored in a clean environment to prevent any
accumulation of dust or other contaminants. Store the glassware inverted or capped with
aluminum foil.
3.2 Interferences co-extracted from the samples will vary considerably from source to source,
depending on the diversity of the industrial complex or municipality being sampled. While
general cleanup procedures are provided as part of this method, unique samples may require
additional cleanup approaches to achieve the detection limits listed in Tables 1 and 2.
4. ‘SAFETY
4.1 The toxicity or carcinogenicity of each reagent used in this method has not been precisely defined;
however, each chemical compound should be treated as a potential health hazard. From this
viewpoint, exposure to these chemicals must be reduced to the lowest possible level by whatever
means available. The laboratory is responsible for maintaining a current awareness file of OSHA
regulations regarding the safe handling of the chemicals specified in this method. A reference file
of material data handling sheets should also be made available to all personnel involved in the
chemical analysis. Additional references to laboratory safety are available and have been
identified 24 for the information of the analyst.
5. APPARA TUS AND EQUIPMENT
5.1 Sample containers: Narrow-mouth glass bottles, 1-L or 1-quart volume, equipped with
pólytetrafluoroethylene (PTFE)-lined screw-caps. Wide-mouth glass bottles, 1-quart volume,
equipped with PIPE-lined screw-caps may also be used. Prior to use, wash bottles and cap liners
with detergent and rinse with tap and distilled water. Allow the bottles and cap liners to air dry,
then muffle at 400°C for 1 hour. After cooling, rinse the bottles and cap liners with hexane, seal
the bottles, and store in a dust-free environment.
604
-------
Method 645
5.1.1 Automatic sampler (optional): Must incorporate glass sample containers for the
collection of a minimum of 250 mL. Sample containers must be kept refrigerated at 4°C
and protected from light during compositing. If the sampler uses a peristaltic pump, a
minimum length of compressible silicone rubber tubing may be used. Before use,
however, the compressible tubing should be thoroughly rinsed with methanol, followed
by repeated rinsings with reagent water to minimize the potential for contamination of
the sample. An integrating flow meter is required to collect flow-proportional
composites.
5.2 Kuderna-Danish (K-D) glassware.
5.2.1 Synder column: Three-ball macro (Kontes K-503000-0121 or equivalent).
5.2.2 Concentrator tube: l0-mL, graduated (Kontes K-570050-1025 or equivalent) with
ground-glass stopper.
5.2.3 Evaporative flask: 500-mL (Kontes K-570001-0500 or equivalent). Attach to
concentrator tube with springs.
5.3 Gas chromatography system.
5.3.1 The gas chromatograph must be equipped with a glass-lined injection port compatible
with the detector to be used. A data system is recommended for measuring peak areas.
5.3.1.1 Column 1: 180cm long by 2mm ID, glass, packed with 10% OV-11 on Gas
Chrom W-HP (100/120 mesh) or equivalent.
5.3.1.2 Column 2: 180 cm long by 2 mm ID, PyrexR glass, packed with 3% Dexsil
300 on Chromasorb W-HP (80/100 mesh) or equivalent.
5.3.1.3 Column 3: 180 cm long by 2mm ID Glass, packed with 3% SP-2100 on
Supelcoport (100/120 mesh) or equivalent.
5.3.1.4 Column 1 was used to develop the accuracy and precision statements in
Section 12. Guidelines for the use of alternative column packings are
provided in Section 10.3.1.
5.3.1.5 Detector: Nitrogenlphosphorous. This detector has proven effective in the
analysis of wastewaters for the parameters listed in Section 1.1 and was used
to develop the method performance statements in Section 12. Guidelines for
the use of alternate detectors are provided in Section 10.3.
5.4 Chromotographic column: 300 mm long by 10 mm ID Chromaflex, equipped with coarse-fitted
bottom plate and PTFE stopcock. (Kontes K-420540-0213 or equivalent).
5.5 Drying column: Approximately 400 nun long by 20 mm ID borosilicate glass, equipped with
coarse-fitted bottom plate.
Balance: Analytical, capable of accurately weighing to the nearest 0.0001 g.
Separatory funnel: 2-L, equipped with PTFE stopcock.
5.6.3 Water bath: Heated with concentric ring cover, capable of temperature control (± 2°C).
The bath should be used in a hood.
5.6.4 Standard solution storage containers: 15-mL bottles with PTFE-Iined screw-caps.
5.6 Miscellaneous.
5.6.1
5.6.2
605
-------
Method 645
5.6.5 BoWing chips: Approximately 10/40 mesh. Heat to 400°C for 30 minutes or perform
a Soxhiet extraction with methylene chloride.
6. REAGEN7S AND COIVSUMABLE MA TEP ALS
6.1 Reagents.
6.1.1 Acetone, bexane, and methylene chloride: demonstrated to be free of analytes.
6.1.2 Florisil: PR grade (60/100 mesh). Purchase activated at 675°C and store in glass
containers with glass stoppers or foil-lined screw-caps. Before use, activate each batch
overnight at 200°C in foil-covered glass containers. To prepare for use, place the
amount necessary for the number of columns to be run in a 500-mL reagent bottle and
add 2% by weight of reagent water. Seal and mix thoroughly by shpking or rolling for
10 minutes. Allow to stand for at least 2 hours prior to use. The mixture must be
homogeneons. Keep the bottle tightly sealed to ensure proper activity.
6.1.3 Reagent water: reagent water is defined as a water in which an interferent is not
observed at the method detection limit of each parameter of interest.
6.1.4 Sodium hydroxide (NaOH) solution (iON): Dissolve 40 g NaOH in reagent water and
dilute to 100 niL.
6.1.5 Sodium sulfate: Granular, anhydrous. Condition by heating at 400°C for 4 hours in a
shallow tray.
6.1.6 Sulfuric acid (11250) solution (1+ 1): Add a measured volume of concentrated 112504
to an equal volume of reagent water.
6.1.7 Sodium thiosulfate ACS, milar.
6.2 Standard stock solutions (1.00 ig/gLL): These solutions may be purchased as certified solutions
or prepared from pure standard materials using the following procedures.
6.21 Prepare standard stock solutions by accurately weighing about 0.0 100 g of pure material.
Dissolve the material in hexane or other suitable solvent and dilute to volume in a 1O-mL
volumetric flask. Larger volumes can be used at the convenience of the analyst. If
oompoundpurityiscertifledat96%orgreater,theweightcanbeusedwithoutcorrection
to calculate the concentration of the standard stock.
6.2.2 Store standard stock solutions at 4°C in l5-mL bottles equipped with PTFE-Iined
screw-caps. Standard stock solutions should be checked frequently for signs of
degradation or evaporation, especially just prior to preparing calibration standards from
th
6.2.3 Standard stock solutions must be replaced after 6 months, or sooner if comparison with
check standards indicates a problem.
7. SAMPlE COLLECTION PRESERVATION, AND STORAGE
7.1 Collect all nçles in duplicate. Grab s2mples must be collected in glass containers.
Conventional sampling practices should be followed, except that the bottle must not be prewashed
with sample before collection.
7.2 The samples must be iced or refrigerated at 4°C from the time of collection until extraction.
606
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Method 645
7.3 Chemical preservatives should not be used in the field unless more than 24 hours will elapse
before delivery to the laboratory. If the samples will not be extracted within 48 hours of
collection, the sample should be adjusted to a pH range of 6.0 to 8.0 with sodium hydroxide or
sulfuric acid.
7.4 All samples must be extracted within 7 days and completely analyzed within 40 days of
extraction 6 .
8. CAIJBRA liON AND STANDARDIZATION
8.1 Calibration.
8.1.1 A set of at least three calibration solutions containing the method analytes is needed. One
calibration solution should contain each analyte at a concentration approaching but greater
than the estimated detection limit (Tables 1 and 2) for that compound; the other two
solutions should contain analytes at concentrations that bracket the range expected in
samples. For example, if the detection limit for a particular analyte is 0.2 j g/L, and a
sample expected to contain approximately 5 g/L is analyzed, solutions of standards
should be prepared at concentrations representing 0.3 &g/L, 5 gIL, and 10 jig/L for the
particular analyte.
8.1.2 To prepare a calibration solution, add an appropriate volume of a standard stock solution
to a volumetric flask and dilute to volume with hexane.
81.3 Starting with the standard of lowest concentration, analyze each calibration standard
according to Section 10.3 and tabulate peak height or area response versus the mass of
analyte injected. The results can be used to prepare a calibration curve for each
compound. Alternatively, if the ratio of response to concentration (calibration factor) is
a constant over the working range (< 10% relative standard deviation), linearity through
the origin can be assumed and the average ratio or calibration factor can be used in place
of a calibration curve.
8.1.4 The working calibration curve or calibration factor must be verified on each working day
by the measurement of one or more calibration standards. If the response for any analyte
varies from the predicted response by more than ± 10%, the test must be repeated using
a fresh calibration standard. If the results still do not agree, generate a new calibration
curve.
9. QUAUTY CONTROL
9.1 Monitoring for interferences.
9.1.1 Analyze a laboratory reagent blank each time a set of samples is extracted. A laboratory
reagent blank is a 1-L aliquot of reagent water. If the reagent blank contains a reportable
level of any analyte, immediately check the entire analytical system to locate and correct
for possible interferences and repeat the test.
9.2 Assessing accuracy.
9.2.1 After every ten samples, and preferably in the middle of each day, analyze a laboratory
control standard. Calibration standards may not be used for accuracy assessments and
the laboratory control standard may not be used for calibration of the analytical system.
607
-------
Method 645
9.2.1.1 Laboratory control standard concentrate: From stock standards prepared as
described in SectiOn 6.3, prepare a laboratory control standard concentrate
that co, inc each analyte of interest at a concentration of 2 g/mL in acetone
or other suitable solvent.
9.2.1.2 Laboratory control standard: Using a pipette, add 1.00 mL of the laboratory
control standard concentrate to a 1-L aliquot of reagent water.
9.2.1.3 Analyze the laboratory control standard as described in Section 10. For each
analyte in the laboratory control standard, calculate the percent recovery (Pt)
with the equation:
Equatloni
1S 1
.
Me
S 1 a The
T, a The
analytical
bso z
results from the laboratoiy
concenmitlon qf the spike, In
control
,igIL
standard,
in
igIL
9.2.2 At least annually, the laboratory should participate in formal performance evaluation
studies, where solutions of unknown concentrations are analyzed and the performance of
all participants is compared.
9.3 Assessing piecli.
9.3.1 PrecIsion sssessmei*s lbr this method are based upon the analysis of field duplicates
(Sect 7.1). Analyze both sample bottles for at least 10% of all samples. To the extent
practical, the samples for duplication should contain reportable levels of most of the
an—.
9.3.2 For each analyte in each duplicate pair, calculate the relative range 7 (RRO with the
equation:
Equatlcn2
100R,
RR =
X i
n*ere
= The
= The
absolute
awrage
difference between the duplicate
concentration finuid in
nts
X
and
X ,
in
igIL
ineasureme
gIL
9.3.3 Individual relative range measurements are pooled to determine average relative range
or to develop an expression of relative range as a function of concentration.
608
-------
Method 645
10. PROCEDURE
10.1 Sample extraction.
10.1.1 Mark the water meniscus on the side of the sample bottle for later determination of
sample volume. Pour the entire sample into a 2-liter separatory funnel. Check the pH
of the sample with wide-range pH paper and adjust to within the range of 5 to 9 with
sodium hydroxide or sulfuric acid.
10.1.2 Add 60 mL of methylene chloride to the sample bottle and shake for 30 seconds to rinse
the walls. Transfer the solvent to the separatory funnel and extract the sample by shaking
the funnel for 2 minutes with periodic venting to release vapor pressure. Allow the
organic layer to separate from the water phase for a minimum of 10 minutes. If the
emulsion interface between layers is more than one-third the volume of the solvent layer,
the analyst must employ mechanical techniques to complete the phase separation. The
optimum technique depends on the sample, but may include stirring, filtration of the
emulsion through glass wool, or centrifugation. Collect the extract in a 250-mL
Erlenmeyer flask.
10.1.3 Add an additional 60-mL volume of methylene chloride to the sample bottle and complete
the extraction procedure a second time, combining the extracts in the Erlenmeyer flask.
10.1.4 Perform a third extraction in the same manner. Pour the combined extract through a
drying column containing about 10 cm of anhydrous sodium sulfate, and collect it in a
500-mL K-D flask equipped with a 10 ml. concentrator tube. Rinse the Erlemneyer flask
and column with 20 to 30 mL of methylene chloride to complete the quantitative transfer.
10.1.5 Add one or two clean boiling chips to the flask and attach a three-ball Snyder column.
Prewet the Snyder column by adding about 1 mL of methylene chloride to the top. Place
the K-D apparatus on a hot water bath (80 to 85°C) so that the concentrator tube is
partially immersed in the hot water and the entire lower rounded surface of the flask is
bathed in steam. Adjust the vertical position of the apparatus and the water temperature
as required to complete the concentration in 15 to 20 minutes. At the proper rate of
distillation, the balls of the column will actively chatter but the chambers will not flood.
When the apparent volume of liquid reaches 1 mL, remove the K-D apparatus and allow
it to drain and cool for at least 10 minutes. Remove the Snyder column and rinse the
flask and its lower joint into the concentrator tube with 1 to 2 mL of methylene chloride.
10.1.6 For Florisil column cleanup or gas chromatography, the extract must be in hexane
solution. To exchange the solvent to hexane, add one or two fresh boiling chips to the
flask and ampule containing the extract, add 50 mL of hexane, and reattach the Snyder
column. Pour about I mL of bexane into the top of the Snyder column, and concentrate
the extract at 85 to 95°C in the hot water bath as above. When the apparent volume of
liquid reaches 1 mL, remove the K-D apparatus from the water bath and allow it to drain
and cool for at least 10 minutes.
101.7 Remove the Snyder column, rinse the flask and its lower joint into the concentrator tube
with 1 to 2 mL of hexane. A 5-mL syringe is recommended for this operation. Dilute
to 10 mL with hexane for analysis by gas chromatography (Section 10.3) if cleanup is
not required. If the extract requires cleanup, proceed to Section 10.2. If the extracts will
609
-------
Method 645
be stored longer than 2 days, they should be transferred to PTFE-sealed screw-cap
bottles. Proceed with gas chromatographic analysis.
10.1.8 Determine the original sample volume by refilling the sample bottle to the mark and
transferring the liquid to a 1,000-mL graduated cylinder. Record the sample volume to
the nearest 5 inL.
10.2 Cleanup and separation.
10.2.1 Cleanup procedures may not be necessary for a relatively clean sample matrix. The
cleanup procedures recommended in this method have been used for the analysis of
alachior, butachior, diph mid, and lethane in various clean waters and municipal
effluents. The use of Florisil as the cleanup material for fluridone and norfiurazon has
been demonstrated to yield recoveries of less than 50%, and is not recommended as a
cleanup material for these compounds. Use of specific detectors may obviate the
necessity for cleanUp of relatively clean sample matrices. If particular circumstances
demand the use of an alternative cleanup procedure, the analyst must determine the
elution profile and demonstrate that the recovery of each compound of interest is no less
than 85%.
10.2.2 Place the necessary amount of deactivated FlorisilR into a 20 mm ID chromatographic
column and tap the column to settle the FlorisilR. Add 1 to 2 cm of anhydrous sodium
sulfate to the top of the FlorisilR.
10.2.3 Pre-elute the column with 50 to 60 mL of hexane. Discard the eluate and, just prior to
exposure of the sodium sulfate layer to the air, transfer the sample extract onto the
column by decantation. Complete the transfer by rinsing with two additional 2-mL
volumes of hexane. Alternatively, a measured aliquot of the extract may be taken for
cleanup.
10.2.4lustpriorto exposureofthesodiumsulfatelayertothe air, elutethe column with 100
mL hexane. Discard the eluate and repeat the elution with 200 mL of 6% acetone in
hexane (WV). Collect the eluate in a 500-mL K-D flask equipped with a 10-niL
concentrator tube (Fraction 1). All elutions should be carried out using a flow rate of
about 5 mL/min.
10.2.5 Perform a second elution with 200 mL of 15% acetone in hexane (Fraction 2). Collect
each fraction in a separate K-D apparatus. The elution pattern for these compounds is
shown in Table 3.
10.2.6 Determine, from Table 3, the fractions of interest and concentrate by standard K-D
technique, as indicated in Section 10.1.5, using hexane in place of methylene chloride,
to avolumeof 10 mL.
10.2.7 Analyze the fractions by gas chromatography.
10.3 Gas chromatography analysis.
10.3.1 Recommended columac and detectors and operating conditions for the gas
chromatography system are described in Section 5.3. Tables I and 2 summarize the
recommended operating conditions for the gas chromatograph. Included in these tables
are retention times and estimated detection limits that can be achieved by this method.
Examples of the separations achieved are shown in Figures 1 through 3. Other packed
column c, chromatographic conditions, or detectors may be used if data quality
£lfl
-------
Method 645
comparable to Table 4 is achieved. Capillary (open-tubular) columns may also be used
if the relative standard deviations of responses for replicate injections are demonstrated
to be less than 6% and data quality comparable to Table 4 is achieved.
10.3.2 Inject 2 to 5 1 iL of the sample extract using the solvent-flush technique. 8 Record the
volume injected to the nearest 0.05 L, the total extract volume, and the resulting peak
size in area or peak height units.
10.3.3 The width of the retention-time window used to make identifications should be based
upon measurements of actual retention-time variations of standards over the course of the
day. Three times the standard deviation of a retention time for a compound can be used
to calculate a suggested window size; however, the experience of the analyst should
weigh heavily in the interpretation of chromatograms.
10.3.4 If the response for the peak exceeds the working range of the system, dilute the extract
and reanalyze.
10.3.5 If the measurement of the peak response is prevented by the presence of interferences,
further cleanup is required.
11. CALCULATiONS
11.1 Determine the concentration (C) of individual compounds in the sample in micrograms per liter
with the equation:
Equation 3
Concentration, zgIL =
where
A = Amount of material injected, in ng
= Voiwne of extract injected, in pL
Vi = Voiwne of total extract, in pL
1’, = Voiwne of water extracted, in mL
11.2 Report the results for the unknown samples in micrograms per liter. Round off the results to the
nearest 0.1 ig/L or two significant figures.
12. METhOD PERFORMANCE
12.1 Estimated detection limits (EDL) and associated chromatographic conditions are listed in Table 1.
The detection limits were calculated from the minimum detectable response of the EC detector
equal to 5 times the GC background noise, assuming a 10-mL final extract volume of a 1-L
sample and a GC injection of 5 tL.
12.2 Single-laboratory accuracy and precision studies were conducted by Environmental Science and
Engineering, 6 using spiked samples. The results of these studies are presented in Table 2.
611
-------
Method 645
13. GC/MS C0AnRMA nON
13.1 It is recommended that GC/MS techniques be judiciously employed to support qualitative
identifications made with this method. The mass spectrometer should be capable of scanning the
mass range from 35 amu to a mass 50 amu above the molecular weight of the compounds of
interest. The instrument must be capable of scanning the mass range at a rate to produce at least
5 scans per peak, but not to exceed 7 scans per peak utilizing a 70 V (nominal) electron energy
in the electron impact ionization mode. A GC to MS interface constructed of all glass or
glass-lined materials is recommended. A computer system should be interfaced to the mass
spectrometer that allows the continuous acquisition and storage on machine-readable media of all
mass spectra obtained throughout the duration of the chromatographic program.
13.2 Gas chromatographic colnmns and conditions should be selected for optimum separation and
performance. The conditions selected must be compatible with standard GCIMS operating.
practices. Chromatographic tailing factors of less than 5.0 must be achieved. The calculation of
tailing factors is illustrated in Method 625.’°
13.3 At the beginning of each day that confirmatory analyses are to be performed, the GC/MS system
must be checked to see that all DFTPP performance criteria are achieved. 11
13.4 To confirm an identification of a compound, the background-corrected mass spectrum of the
compound must be obtained from the sample extract and compared with a mass spectrum from
a stock or calibration standard analyzed under the same chromatographic conditions. It is
recommended that at least 25 ng of material be injected into the GCIMS. The criteria below must
be met for qualitative confirmation.
13.4.1 The molecular ion and other ions that are present above 10% relative abundance in the
m ssspectrumofthestandardmustbepresentinthemass spectrumofthesamplewith
agreement to ± 10%. For example, if the relative abundance of an ion is 30% in the
m s spectrum of the standard, the allowable limits for the relative abundance of that ion
in the mass spectrum for the sample would be 20 to 40%.
13.4.2 Theretentiontimeofthecompound inthesamplemustbewithin6 seconds ofthesame
compound in the standard solution.
13.4.3 Compounds that have similar mass spectra can be explicitly identified by GCIMS only
on the basis of retention time data.
13.5 Where available, chemical ionization mass spectra may be employed to aid in the qualitative
identification process.
13.6 Should these MS procedures fail to provide satisfactory results, additional steps may be taken
before reanalysis. These may include the use of alternative packed or capillary GC columns or
additional cleanup.
cs-s
-------
Method 645
References
1. ASTM Annual Book of Standards, Part 31, D3694, “Standard Practice for Preparation of Sample
Containers and for Preservation,” American Society for Testing and Materials, Philadelphia,
Pennsylvania, p. 679, 1980.
2. “Carcinogens - Working with Carcinogens,” Department of Health, Education, and Welfare,
Public Health Service, Center for Disease Control, National Institute for Occupational Safety and
Health, Publication No. 77-206, Aug. 1977.
3. “OSHA Safety and Health Standards, General Industry” (29 CFR 1910), Occupational Safety and
Health Administration, OSHA 2206 (Revised, January 1976).
4. “Safety in Academic Chemistry Laboratories,” American Chemical Society Publication,
Committee on Chemical Safety, 3rd Edition, 1979.
5. ASTM Annual Book of Standards, Part 31, D3370, “Standard Practice for Sampling Water,”
American Society for Testing and Materials, Philadelphia, Pennsylvania, p. 76, 1980.
6. Test procedures for Pesticides in Wastewaters, EPA Contract Report #68-03-2897. Unpublished
report available from U.S. Environmental Protection Agency, Environmental Monitoring and
Support Laboratory, Cincinnati, Ohio.
7. “Handbook for Analytical Quality Control in Water and Wastewater Laboratories,”
EPA-600/4-79-019, U.S. Environmental Protection Agency, Environmental Monitoring and
Support Laboratory - Cincinnati, Ohio, March 1979.
8. Burke, J.A., “Gas Chromatography for Pesticide Residue Analysis; Some Practical Aspects,”
Journal of the Association of Official Analytical Chemists, 48, 1037 (1965).
9. “Evaluation of Ten Pesticide Methods,” U.S. Environmental Protection Agency, Contract No.
68-03-1760, Task No. 11, U.S. Environmental Monitoring and Support Laboratory, Cincinnati,
Ohio (In preparation).
10. “MethOdS for Organic Chemical Analysis of Municipal and Industrial Wastewater”
EPA-600/4-82-057, U.S. Environmental Protection Agency, Environmental Monitoring and
Support Laboratory - Cincinnati, Ohio.
11. Eichelberger, J.W., Harris, L.E., and Budde, W.L., Anal. Chem., 46, 1912 (1975).
613
-------
Method 645
Table 1. Gas Chromatography and Detection Limits of Certain Amines and
Lethane
Estimated
Parameter Retention rime (ma ,) Detection Limit
Column 1 Column 2 Column 3 (pg/Li
Alachlor 6.9 0.2
Butachlor 10.5 0.3
Diphenamide 10.8 0.2
Fluridone 2.2 2.45 2.1 0.5
Lethane 2.0 0.1
Norfiurazon 18.4 0.02
Column 1:180 cm long by 2 mm ID glass, packed with 10% OV-11 on Gas Chrom W-HP, 100/120
mesh; nitrogen carrier gas at a flow rate of 30 mL/min. Column temperature is held at 225°C for 4
minutes after injection and then programmed to 275°C at 4°/mm and held for 8 minutes.
CoLumn 2:180 cm long by 2 mm ID glass, packed with 3% Dexsil 300 on Chromasorb W-HP, 80/100
mesh; nitrogen carrier gas at a flow rate of 30 mL/min. Column temperature at 300°C isothermal.
Column 3:180 cm long by 2 mm ID glass, packed with 3% SP-2 100 on Supelcoport, 100/120 mesh;
nitrogen carrier gas at a flow rate of 40 mL/min. Column temperature at 275°C isothermal.
614
-------
Method 645
Table 2. Single-Laboratory Accuracy and Precision
Average Relative Standard
Spike Range No. of Percent Deviation (96)
Parameter Matrix Type (pg/Li Replicates Recovery
Alachlorn 1 255 7 113 9.0
1 996 7 104 13.3
Butachior 1 286 7 93.1 8.2
1 1,420 7 92.8 4.3
Diphenamid 2 9.3 7 100 14.2
3 740 7 98.8 7.0
Fluridone 1 20.8 7 92.0 11.5
1 998 7 88.4 11.4
Lethane 1 167 7 93.3 19.9
1 576 7 97.6 29.4
Nor1urazone 1 243 7 89.5 7.4
3 1,048 7 102 6.1
* I = Manufacturing effluent wastewateIS.
2 = Manufacturing effluent wastewater + POTW effluent at a ratio of 1:200.
3 = Manufacturing effluent wastewater + POTW effluent at a ration of 1:1.
Florisil cleanup not employed.
Table 3. Florisil’ Cleanup Recoveries
Solvent Average Percent Recoveries
F,action ” Ala chior ButachiOr Dip henamid Lethane
1 103 95 106
2 ND ND 96 ND
2% deactivated.
1 = 6% acetone/hexane
2 = 15% acetone/hexafle
515
-------
Retentlon lime In parentheses
Figure 1. Gas Chromatogram of Amines/Lethane (Column 2).
Lethane (2.O)
Alachior (6.9)
Butachior (1O.5)
/ Nortlurazon
A -C -73
616
-------
Method 645
Flundone
7,
0 5
Retention Time (minutes)
A52-002-74
Figure 2. Gas Chromatogram of Fluridofle (Column 2).
617
-------
MeThod 645
Fkiridone
/
I
0 2.0 4.0
R .ntIon Tim. (minutes)
A C -75
Figure 3. Gas Chromatogram of Flundone (Column 3).
618
-------
Method 646
The Determination of Din itro
Aromatic Pesticides in
Municipal and Industrial
Waste water
-------
Method 646
The Determination of Dinitro Aromatic Pesticides in Municipal and
Industrial Wastewater
1. SCOPE AND APPLICA TION
1.1 This method covers the determination of certain dinitro aromatic pesticides in municipal and
industrial wastewater. The following parameters may be determined by this method.
Parameter CAS No.
Basalin (Fluchioralin) 33245-39-5
CDN 97-00-7
Dinocap 39300-45-3
1.2 The estimated detection limit (EDL) for each parameter is listed in Table 1. The EDL was
calculated from the minimum detectable response of the electron capture detector (ECD) equal to
5 times the GC background noise assuming a 1.0 mL final extract volume of a 1-L reagent water
sample and a GC injection of 5 L. The EDL for a specific wastewater may be different
depending on the nature of interferences in the sample matrix.
1.3 This is a gas chromatographic (GC) method applicable to the determination of the compounds
listed above in municipal and industrial discharges. When this method is used to analyze
unfamiliar samples for any or all of the compounds listed above, compound identifications should
be supported by at least one additional qualitative technique. Section 13 provides gas
chromatograph/mass spectrometer (GC/MS) conditions appropriate for the qualitative confirmation
of compound identifications.
1.4 This method is restricted to use by or under the supervision of analysts experienced in the
operation of gas chromatographs and in the interpretation of chromatograms.
2. SUMMARY OF METHOD
2.1 Dinitroaromatic pesticides are removed from the sample matrix by extraction with 15% methylene
chloride in hexane. The extract is dried, exchanged into hexane, and analyzed by gas
chromatography (GC). Column chromatography is used as necessary to eliminate interferences
which may be encountered. Measurement of the pesticides is accomplished with an electron
capture detector.
2.2 Confirmatory analysis by gas chromatography/mass spectrometry (GC/MS) is recommended
(Section 13) when a new or undefined sample type is being analyzed, if the concentration is
adequate for such determination.
3. INTERFERENCES
3.1 Solvent, reagents, glassware, and other sample-processing hardware may yield discrete artifacts
and/or elevated baselines causing misinterpretation of gas chromatograms. All of these materials
must be demonstrated to be free from interferences under the conditions of the analysis by running
laboratory reagent blanks as described in Section 9.1.
621
-------
Method 646
3.1.1 The use of high-purity reagents and solvents helps to minimize interference problems.
Purification of solvents by distillation in all-glass systems may be required.
3.1.2 Glassware must be scrupulously cleaned.’ Clean all glassware as soon as possible after
use by rinsing with the last solvent used in it. This should be followed by detergent
washing with hot water and rinses with tap water and reagent water. It should then be
drained dry and heated in a muffle furnace at 400°C for 15 to 30 minutes. Solvent rinses
with acetone and pesticide-quality hexane may be substituted for the heating. Volumetric
ware should not be heated in a muffle furnace. After drying and cooling, glassware
should be sealed and stored in a clean environment to prevent any accumulation of dust
or other Coi min nts . Store the glassware inverted or capped with aluminum foil.
3.2 Interferences coextracted from the samples will vary considerably from source to source,
depending on the diversity of the industrial complex or municipality being sampled. While
general cleanup procedures are provided as part of this method, unique samples may require
additional cleanup approaches to achieve the detection limits listed in Table 1.
4. SAFETY
4.1 The toxicity or carcinogenicity of each reagent used in this method has not been precisely defined;
however, each chemical compound should be treated as a potential health hazard. From this
viewpoint, exposure to these chemicals must be reduced to the lowest possible level by whatever
means available. The laboratory is responsible for maintaining a current awareness file of OSHA
regulations regarding the safe handling of the chemicals specified in this method. A reference file
of material data handling sheets should also be made available to all personnel involved in the
chemical analysis. Additional references to laboratory safety are available and have been
identified 2 ’ for the information of the analyst.
5. APPAR4 TUS AND EQWPMENT
5.1 Sample containers: Narrow-mouth glass bottles, 1-L or 1-quart volume, equipped with
polytetrafluoroethylene (PTFE)-lined screw-caps. Wide-mouth glass bottles, 1-quart volume,
equipped with P’fl E-lined screw-caps may also be used. Prior to use, wash bottles and cap liners
with detelgent and rinse with tap and distilled water. Allow the bottles and cap liners to air dry,
then muffle at 400°C for 1 hour. After cooling, rinse the cap liners with hexane, seal the bottles,
afld store in a dust-free environment.
5.1.1 Automatic sampler (optional): Must incorporate glass sample containers for the
collection of a minimum of 250 mL. Sample containers must be kept refrigerated at 4°C
and protected from light during compositing. If the sampler uses a peristaltic pump, a
minimum length of compressible silicone rubber tubing may be used. Before use,
however, the compressible tubing should be thoroughly rinsed with methanol, followed
by repeated rinsings with reagent water to minimize the potential for contamination of
the sample. An integrating flow meter is required to collect flow-proportional
composites.
5.2 Kuderna-Danish (X-D) glassware.
5.2.1 Synder column: Three-ball macro (Kontes K-503000-0121 or equivalent) and two-ball
micro (Kontes K-569001-0219 or equivalent).
622
-------
Method 646
5.2.2 Concentrator tube: 1O-mL, graduated (Kontes K-570050-1025 or equivalent) with
ground-glass stopper.
5.2.3 Evaporative flask: 500-mL (Kontes K-570001-0500 or equivalent). Attach to
concentrator tube with springs.
5.3 Gas chromatography system.
5.3.1 The gas chromatograph must be equipped with a glass-lined injection port compatible
with the detector to be used. A data system is recommended for measuring peak areas.
5.3.1.1 Chromatography column: 180 cm long by 4 mm ID, glass, packed with
1.5% OV-1711.95% OV-210 on Supelcoport (100/120 mesh) or equivalent.
This column was used to develop the method performance statements in
Section 12. Guidelines for the use of alternative column packings are
provided in Section 10.3.1.
5.3.1.2 Detector: Electron capture. This detector has proven effective in the analysis
of wastewaters for the parameters listed in the scope and was used to develop
the method performance statements in Section 12. Guidelines for the use of
alternative detectors are provided in Section 10.3.
5.4 Chromatographic column: 200 mm long by ‘10 mm ID Chromaflex, equipped with coarse-fitted
bottom plate and PTFE stopcock. (Kontes K-420540-0213 or equivalent).
5.5 Drying column: Approximately 400 mm long by 20 mm ID borosilicate glass, equipped with
coarse-fitted bottom plate.
5.6 Miscellaneous
5.6.1 Balance: analytical, capable of accurately weighing to the nearest 0.0001 g.
5.6.2 Separatory funnel: 2-L, equipped with PTFE stopcock.
5.6.3 Water bath: heated, with concentric ring cover, capable of temperature control (± 2°C).
The bath should be used in a hood.
5.6.4 Standard solution storage containers: 15-mL bottles with PTFE-lined screw-caps.
6. REAGENTS AND CONSUMABLE MA TERIALS
6.1 Reagents.
6.1.1 Acetone, hexane, and methylene chloride: Demonstrated to be free of analytes.
6.1.2 Florisil: PR grade (60/100 mesh). Purchase activated at 675°C and store in dark in
glass containers with glass stoppers or foil-lined screw-caps. Before use, activate each
batch overnight at 200°C in foil-covered glass container.
6.1.3 Reagent water: Reagent water is defined as a water in which an interferent is not
observed at the method detection limit of each parameter of interest.
6.1.4 Sodium hydroxide (NaOH) solution (iON): Dissolve 40 g NaOH in reagent water and
dilute to 100 mL.
6.1.5 Sodium sulfate: Granular, anhydrous. Condition by heating at 400°C for 4 hours in a
shallow tray.
623
-------
Method 646
6.1.6 Sulfuric acid (11250) solution (1+1): Add measured volume of concentrated H 2 S0 4 to
equal volume of reagent water.
6.2 Standard stock solutions (1.00 pg/pL): These solutions may be purchased as certified solutions
or prepared from pure standard materials using the following procedures.
6.2.1 Prepare standard stock solutions by accurately weighing about 0.0100 g of pure material.
Dissolve the material in hexane and dilute to volume in a 10-niL volumetric flask.
Larger volumes can be used at the convenience of the analyst. If compound purity is
certified at 96% or greater, the weight can be used without correction to calculate the
concentration of the standard stock.
6.2.2 Store standard stock solutions at 4°C in 15-mL bottles equipped with PTFE-lined
screw-caps. Standard stock solutions should be checked frequently for signs of
degradation or evaporation, especially just prior to preparing calibration standards from
them.
6.2.3 Standard stock solutions must be replaced after 6 months, or sooner if comparison with
check standards indicates a problem.
7. SAMPLE CouEcnoN, PRESERVATiON, AND STORAGE
7.1 Collect all samples in duplicate. Grab samples must be collected in glass-containers.
Conventional sampling practices 3 should be followed, except that the bottle must not be prewashed
with sample before collection.
7.2 The samples must be iced or refrigerated at 4°C from the time of collection until extraction.
7.3 All samples must be extracted within 30 days of collection. 6
8. CALIBRATiON AND STANDARDIZATiON
8.1 Calibration.
8.1.1 A set of at least three calibration solutions containing the method analytes is needed. One
calibration solution should contain each analyte at a concentration approaching but greater
than the estimated detection limit (Table 1) for that compound; the other two solutions
should contain analytes at concentrations that bracket the range expected in samples. For
example, if the detection limit for a particular analyte is 0.2 g/L, and a sample expected
to contain approximately 5 ,ug/L is analyzed, standard solutions should be prepared at
concentrations of 0.3 g/L, 5 gIL, and 10 ig/L.
8.1.2 To prepare a calibration solution, add an appropriate volume of a standard stock solution
to a volumetric flask and dilute to volume with hexane.
8.1.3 Starting with the standard of lowest concentration, analyze each calibration standard
according to Section 10.3 and tabulate peak height or area responses versus the mass of
analyte injected. The results can be used to prepare a calibration curve for each
compound. Alternatively, if the ratio of response to concentration (calibration factor) is
a constant over the working range (<10% relative standard deviation), linearity through
the origin can be assumed and the average ratio or calibration factor can be used in place
of a calibration curve.
624
-------
Method 646
8.1 .4 The working calibration curve or calibration factor must be verified on each working day
by the measurement of one or more calibration standards. If the response for any analyte
varies from the predicted response by more than ± 10%, the test must be repeated using
a fresh calibration standard. If the results still do not agree, generate a new calibration
curve.
8.2 Florisil standardization.
8.2.1 Florisil from different batches or sources may vary in absorptive capacity. To
standardize the amount of Florisil which may be used in the cleanup procedure
(Section 10.2) use of the lauric acid value 7 is suggested. The referenced procedure
determines the adsorption from hexane solution of lauric acid, in milligrams per gram of
Florisil. The amount of Florisil to be used for each column is calculated by dividing this
factor into 110 and multiplying by 20 g.
9. QuALITY CONTROL
9.1 Monitoring for interferences: Analyze a laboratory reagent blank each time a set of samples is
extracted. A laboratory reagent blank is a 1- .L aliquot of reagent water. If the reagent blank
contains a reportable level of any analyte, inunediately check the entire analytical system to locate
and correct for possible interferences and repeat the test.
9.2 Assessing accuracy.
9.2.1 After every ten samples, and preferably in the middle of each day, analyze a laboratory
control standard. Calibration standards may not be used for accuracy assessments and
the laboratory control standard may not be used for calibration of the analytical system.
9.2.1.1 Laboratory control standard concentrate: From stock standards prepared as
described in Section 6.3, prepare a laboratory control standard concentrate
that contains each analyte of interest at a concentration of 2 /2g/mL in acetone
or other suitable solvent. 8
9.2.1.2 Laboratory control standard: Using a pipette, add 1.00 mL of the laboratory
control standard concentrate to a 1-L aliquot of reagent water.
9.2.1.3 Analyze the laboratory control standard as described in Section 10. For each
analyte in the laboratory control standard, calculate the percent recovery (Ps)
with the equation:
Equation 1
1 OOS.
where
S 1 = The analytical results from the laboratory control standard, in g/L
= The known concentration of the spike, in ig/L
625
-------
Method 646
9.2.2 At least annually, the laboratory should participate in formal performance evaluation
studies, where solutions of unknown concentrations are analyzed and the performance of
all participants is compared.
9.3 Assessing precision.
9.3.1 Precision assessments for this method are based upon the analysis of field duplicates
(Section 7.1). Analyze both sample bottles for at least 10% of all samples. To the extent
practical, the samples for duplication should contain reportable levels of most of the
an—.
9.3.2 For each analyte in each duplicate pair, calculate the relative range (RR ) with the
equation:
Equation 2
KR 1 100R,
)4.’zere
= The
= The
absolute
average
dWerence
between the duplicate measurements
n jbwid [ 1 2], in g/L
X 1
and
X 2 ,
in
JLgIL
concentratlo
9.3.3 Individual relative range measurements are pooled to determine average relative range
or to develop an expression of relative range as a function of concentration.
10. PROCEDURE
10.1 Sample extraction.
10.1.1 Mark the water meniscus on the side of the sample bottle for later determination of
sample volume. Pour the entire sample into a 2-L separatory funnel. Check the pH of
the sample with wide-range pH paper and adjust to within the range of 5 to 9 with
sodium hydroxide or sulfuric acid.
10.1.2 Add 60 mL of 15% methylene chioride/hexane to the sample bottle and shake for 30
seconds to rinse the walls. Transfer the solvent to the separatory funnel and extract the
sample by shaking the funnel for 2 minutes with periodic venting to release vapor
pressure. Allow the organic layer to separate from the water phase for a minimum of
10 minutes. If the emulsion interface between layers is more than one-third the volume
of the solvent layer, the analyst must employ mechanical techniques to complete the
phase separation. The optimum technique depends on the sample, but may include
stirring, filtration of the emulsion through glass wool, or centrifugation. Collect the
extract in a 250-mL Erlenmeyer flask.
10.1.3 Add an additional 60-mL volume of 15% methylene chioride/hexane to the sample bottle
and complete the extraction procedure a second time, combining the extracts in the
Erienmeyer flask.
626
-------
Method 646
10.1.4 Perform a third extraction in the same manner. Pour the combined extract through a
drying column containing about 10 cm of anhydrous sodium sulfate, and collect it in a
500-rnL K-D flask equipped with a 10-mL concentrator tube.
10.1.5 Add one or two clean boiling chips to the flask and attach a three-ball Snyder column.
Prewet the Snyder column by adding about 1 mL of hexane to the top. Place the K-D
apparatus on a hot water bath (80 to 85°C) so that the concentrator tube is partially
immersed in the hot water and the entire lower rounded surface of the flask is bathed in
steam. Adjust the vertical position of the apparatus and the water temperature as required
to complete the concentration in 15 to 20 minutes. At the proper rate of distillation, the
balls of the column will actively chatter but the chambers will not flood. When the
apparent volume of liquid reaches 1 mL, remove the K-D apparatus and allow it to drain
and cool for at least 10 minutes. Remove the Snyder column and rinse the flask and its
lower joint into the concentrator tube with I to 2 mL of hexane. A 5-mL syringe is
recommended for this operation. If the extract requires cleanup proceed to Section 10.2
(cleanup and separation). If cleanup has been performed or if the extract does not require
cleanup, proceed with Section 10.1.6.
10.1.6 Add a clean boiling chip to the concentrator tube. Attach a two-ball micro-Snyder
column. Prewet the micro-Snyder column by addingabout 0.5 mL of hexane to the top.
Place this micro K-D apparatus on a steaming-water bath (80 to 85°C) so that the
concentrator tube is partially immersed in the hot water. Adjust the vertical position of
the apparatus and water temperature as required to complete the concentration in 5 to 10
minutes. At the proper rate of distillation, the balls will actively chatter but the chambers
will not flood. When the apparent volume of liquid reaches 0.5 mL, remove the K-D
apparatus and allow it to drain and cool for at least 10 minutes. Remove the
micro-Snyder column and rinse its lower joint into the concentrator tube with a small
volume of hexane. Adjust the final volume to 1.0 mL, and stopper the concentrator tube;
store refrigerated if further processing will not be performed immediately. If the extracts
will be stored longer than 2 days, they should be transferred to PTFE-sealed screw-cap
bottles. Proceed with gas chromatographic analysis.
10.1.7 Determine the original sample volume by refilling the sample bottle to the mark and
transferring the liquid to a l000-mL graduated cylinder. Record the sample volume to
the nearest 5 mL.
10.2 Cleanup and separation.
10.2.1 Cleanup procedures may not be necessary for a relatively clean sample matrix. The
cleanup procedures recommended in this method have been used for the analysis of
various clean waters and municipal effluents. The Florisil cleanup procedure allows for
a select fractionation of the compounds and will eliminate non-polar materials. The
single-operator precision and accuracy data in Table 2 were gathered using the
recommended cleanup procedures. If particular circumstances demand the use of an
alternative cleanup procedure, the analyst must determine the elution profile and
demonstrate that the recovery of each compound of interest is no less than that recorded
in Table 2.
627
-------
Method 646
10.2.2 Prepare a slurry of 10 g of Florisil in methylene chloride. Use it to pack a 10-mm ID
chromatography column, gently tapping the column to settle the Florisil. Add a 1-cm
layer of anhydrous sodium sulfate to the top of the Florisil.
10.2.3 Just prior to exposure of the sodium sulfate layer to the air, transfer the sample extract
onto the column using an additional 2 mL of hexane to complete the transfer.
10.2.4 Just prior to exposure of the sodiwn sulfite layer to the air, add 30 mL of 50%
methylene chioride/hexane and continue the elution of the column. Elution of the column
should be at a rate of about 2 mLlmin. Discard the eluate from this fraction.
10.2.5 Next, elute the column with 30 mL of methylene chloride, collecting the eluate in a
500-mL K-D flask equipped with a l0-mL concentrator tube. Add 50 mL of hexane to
the flask and concentrate the collected fraction by the standard technique prescribed in
Sections 10.1.5 and 10.1.6. This fraction should contain DCN and basalin.
10.2.6 Elute the column with 30 mL of 10% acetonelmethylene chloride collecting the eluate in
a 500-niL K-D flask equipped with a 10 mL concentrator tube. Add 50-mL of hexane
to the flask and concentrate the collected fraction by the standard technique prescribed
in Sections 10.1.5 and 10.1.6. This fraction should contain dinocap.
10.2.7 Analyze the fractions by gas chromatography.
10.3 Gas chromatography analysis.
10.3.1 Recommended columns and detectors for the gas chromatography system are described
in Section 5.3. Table 1 summarizes the recommended operating conditions for the gas
chromatograph. Included in this table are estimated retention times and detection limits
that can be achieved by this method. Examples of the separations achieved are shown
in Figures 1 and 2. Other packed columns, chromatographic conditions, or detectors
may be used if data quality comparable to Table 2 are achieved. Capillary (open-tubular)
columns may also be used if the relative standard deviations of responses for replicate
injections are demonstrated to be less than 6% and data quality comparable to Table 2
are achieved.
10.3.2 Inject 2 to 5 tL of the sample extract using the solvent-flush technique. 9 Record the
volume injected to the nearest 0.05 giL, the total extract volume, and the resulting peak
size in area or peak height units.
10.3.3 The width of the retention-time window used to make identifications should be based
upon measurements of actual retention-time variations of standards over the course of the
day. Three times the standard deviation of a retention time for a compound can be used
to calculate a suggested window size; however, the experience of the analyst should
weigh heavily in the interpretation of chromatograms.
10.3.4 If the response fbr the peak exceeds the working range of the system, dilute the extract
and reanalyze.
10.3.5 If the measurement of the peak response is prevented by the presence of interferences,
further cleanup is required.
628
-------
Method 646
I 1. CALCULATIONS
11.1 Determine the concentration (C) of individual compounds in the sample in micrograms per liter
with the equation:
Equation 3
( A)(V )
Concentration, j g/L = ______
(V 1 )(V 5 )
where
A = Amount of material injected, in ng
V 1 = Voiwne of extract injected, in pL
V Volume of total extract, in ,iL
V, = Volume of water extracted, in niL
11.3 Report the results for the unknown samples in micrograms per liter. Round off the results of the
nearest 0.1 1 igIL or two significant figures.
12. METHOD PERFORMANCE
12.1 Estimated detection limits and associated chromatographic conditions are listed in Table 1.10 The
detection limits were calculated from the minimum detectable response of the ECD equal to 5
times the GC background noise, assuming a 1 .0-rnL final extract volume of a 1-L sample and a
GC injection of 5 JLL.
12.2 Single-laboratory accuracy and precision studies were conducted by Environmental Science and
Engineering, Inc., 6 using spiked wastewater samples. The results of these studies are presented
in Table 2.
13. GC/MS CONFIRMA TFON
13.1 It is recommended that GCIMS techniques be judiciously employed to support qualitative
identifications made with this method. The mass spectrometer should be capable of scanning the
mass range from 35 amu to a mass 50 amu above the molecular weight of the compound. The
instrument must be capable of scanning the mass range at a rate to produce at least 5 scans per
peak, but not to exceed 7 seconds per scan utilizing a 70 V (nominal) electron energy in the
electron impact ionization mode. A ( iC to MS interface constructed of all glass or glass-lined
materials is recommended. A computer system should be interfaced to the mass spectrometer that
allows the continuous acquisition and storage on machine-readable media of all mass spectra
obtained throughout the duration of the chromatographic program.
13.2 Gas chromatographic columns and conditions should be selected for optimum separation and
performance. The conditions selected must be compatible with standard GC/MS operating
practices. Chromatographic tailing factors of less than 5.0 must be achieved. The calculation of
tailing factors is illustrated in Method 625.11
13.3 At the beginning of each day that confirmatory analyses are to be performed, the (ICIMS system
must be checked to see that all DFTPP performance criteria are achieved.’ 2
629
-------
Method 646
13.4 To confirm an identification of a compound, the background-corrected mass spectrum of the
compound must be obtained from the sample extract and compared with a mass spectrum from
a stock or calibration standard analyzed under the same chromatographic conditions. It is
recommended that at least 25 ng of material be injected into the GC/MS. The criteria below must
be met for qualitative confirmation.
13.4.1 The molecular ion and other ions that are present above 10% relative abundance In the
mace spectn!m of the standard must be present in the mass spectrum of the sample with
agreement to ±10%. For example, if the relative abundance of an ion is 30%. in the
mass spectrum of the standard, the allowable limits for the relative abundance of that ion
inthemassspectrumforthesamplewouldbe20to4O%.
13.4.2 The retention time of the compound in the sample must be within seven seconds of the
same compound in the standard solution.
13.4.3 Compounds that have similar macs spectra can be explicitly identified by GC/MS only
on the basis of retention time data.
13.5 Where available, chemical ionization macs spectra may be employed to aid in the qualitative
identification process.
13.6 Should these MS procedures fail to provide satisfactory results, additional steps may be taken
befbre reanalysis. These may include the use of alternative packed or capillary GC columns or
additional cleanup.
630
-------
Method 646
References
1. ASTM Annual Book of Standards, Part 31, 03694, “Standard Practice for Preparation of Sample
Containers and for Preservation,” American Society for Testing and Materials, Philadelphia,
Pennsylvania, p. 679, 1980.
2. “Carcinogens - Working with Carcinogens,” Department of Health, Education, and Welfare,
Public Health Service, Center for Disease Control, National Institute for Occupational Safety and
Health, Publication No. 77-206, Aug. 1977.
3. “OSHA Safety and Health Standards, General Industry” (29 CFR 1910), Occupational Safety and
Health Administration, OSHA 2206 (Revised, January 1976).
4. “Safety in Academic Chemistry Laboratories,” American Chemical Society Publication,
Committee on Chemical Safety, 3rd Edition, 1979.
5. ASTM Annual Book of Standards, Part 31, 03370, “Standard Practice for Sampling Water,”
American Society for Testing and Materials, Philadelphia, Pennsylvania, p. 76, 1980.
6. Test procedures for Pesticides in Wastewaters, EPA Contract Report #68-03-2897 unpublished
report available from the U.S.Environmental Protection Agency, Environmental Monitoring and
Support Laboratory, Cincinnati, Ohio.
7. Mills, P.A., “Variation of Floricil Activity: Simple Method for Measuring Adsorbent Capacity
and Its Use in Standardizing Florisil Columns,” Journal of the Association of Official Analytical
Chemists, 51, 19, 1968.
8. “Handbook for Analytical Quality Control in Water and Wastewater Laboratories,”
EPA-600/4-79-019, U.S. Environmental Protection Agency, Environmental Monitoring and
Support Laboratory, Cincinnati, Ohio 45268, March 1979.
9. Burke, J.A., “Gas Chromatography for Pesticide Residue Analysis; Some Practical Aspects,”
Journal of the Association of Official Analytical Chemilts, 48, 1037(1965).
10. “Evaluation of Ten Pesticide Methods,” U.S. Environmental Protection Agency, Contract
No.68-03-1760, Task No.11, U.S. Environmental Monitoring and Support Laboratory, Cincinnati,
Ohio.
11. “Methods for Organic Chemical Analysis of Municipal and Industrial Wastewater,” U.S.
Environmental Protection Agency, Environmental Monitoring and Support Laboratory, Cincinnati,
Ohio.
12. Eichelberger, J.W., Harris, L.E., and Budde, W.L., Anal. Chem., 46, 1912, 1975.
631
-------
Method 646
Table 1. Chromatographic Conditions and Estimated Detection Limits
Parameter Retention Time Estimated Detection
(mini Limit (pg/LI
CDN 2.0 .0005
Basalin 6.4 .0005
DinoCap* 10-16 0.1
* Oven temperature 200°C isothermal.
Conditions: 180 cm long by 4 mm ID, glass column, packed with 1.5% OV-17/1.95% OV-210 on
Supelcoport (100/120 mesh) or equivalent; 5% methane/95% argon carrier gas at 33 mL/min flow rate.
Oven temperature 160°C isothermaL
Table 2. Single-Laboratory Accuracy and Precision
Spice Average Standard
Matrk Range Number of Percent Deviation
Parameter Type (pg/Li Replicates Recovery (%)
Basalin 1 10 7 79.0 7.0
1 121 7 99.3 10.1
CDN 1 10 7 78.6 7.6
1 99.2 7 99.5 6.1
Dinocap 1 10 7 108.5 4.5
1 161 7 100.3 4.4
*1= Publidy Owned Treatment Works (POTW) wastewater
632
-------
Method 646
0 2.0
Figure 1. 4-Peak Gas Chromatogram of Dinocap.
Dinocap
4.0 6.0 8.0 10.0 12.0 14.0 16.0
Retention Time (minutes)
18.0
A52-00276
633
-------
ffiod 646
CON
/
Basahn
Ralsidlon Time (minutes)
Figure 2. Gas Chromatogram of CDN and Basalin
634
-------
Method 1656
The Determination of
Organo-Halide Pesticides in
Municipal and Industrial
Waste water
-------
Method 1656
The Determination of Organo-Halide Pesticides in Municipal and
Industrial Waste water
SCOPE AND APPLICATION
1.1 This method is designed to meet the survey requirements of the Environmental Protection Agency
(EPA). It is used to determine (1) the organo-halide pesticides and polychiorinated biphenyls
(PCBs) associated with the Clean Water Act, the Resource Conservation and Recovery Act, and
the Comprehensive Environmental Response, Compensation and Liability Act; and (2) other
compounds amenable to extraction and analysis by wide-bore capillary column gas
chromatography (GC) with halogen-specific detectors.
1.2 The compounds listed in Table 1 may be determined in waters, soils, sediments, and sludges by
this method. The method is a consolidation of several EPA wastewater methods. For waters, the
sample extraction and concentration steps are essentially the same as in these methods. However,
the extraction and concentration steps have been extended to other sample matrices. The method
may be applicable to other pesticides as well. The quality control requirements in this method
give the steps necessary to determine this applicability.
1.3 This method is applicable to a large number of compounds. Calibrating the GC systems for all
compounds is time-consuming. If only a single compound or small number of compounds is to
be tested for, it is necessary to calibrate the GC systems and meet the performance specifications
in this method for these compounds only. In addition, the GC conditions can be optimized for
these compounds provided that all performance specifications in this method are met.
1.4 When this method is applied to analysis of unfamiliar samples, compound identity must be
supported by at least one additional qualitative technique. This method describes analytical
conditions for a second gas chromatographic column that can be used to confirm measurements
made with the primary column. Gas chromatography/mass spectrometry (GC/MS) can be used
to confirm compounds in extracts produced by this method when analyte levels are sufficient.
1.5 The detection limits of this method are usually dependent on the level of interferences rather than
instrumental limitations. The limits in Table 2 typify the minimum quantities that can be detected
with no interferences present.
1.6 This method is for use by or under the supervision of analysts experienced in the use of a gas
chromatograph and in the interpretation of gas chromatographic data. Each laboratory that uses
this method must demonstrate the ability to generate acceptable results using the procedure in
Section 8.2.
2. SUMMARY OF METHOD
2.1 Extraction.
2.1.1 The percent solids content of a sample is determined.
2.1.2 Samples containing low solids: If the solids content is 1% or less, a l-L sample is
extracted with methylene chloride using continuous extraction techniques.
637
-------
Method 1656
2.1.3 Samples containing less than 1% solids.
2.1.3.1 Non-sludge samples: If the solids content is 1 to 30%, the sample is diluted
to 1% solids with reagent water, homogenized ultrasonically, and extracted
with methylene chloride using continuous extraction techniques. If the solids
content is greater than 30%, the sample is extracted with methylene
chloride:acetone using ultrasonic techniques.
2.1.3.2 Municipal sludge samples and other intractable sample types: If the solids
content is less than 30%, the sample is diluted to 1% solids and extracted
with methylene chloride using continuous extraction techniques. If the solids
content is greater than 30%, the sample is extracted with acetonitrile and
then methylene chloride using ultrasonic techniques. The extract is back-
extracted with 2% (W/V) sodium sulfate in reagent water to remove water-
soluble interferences and residual acetonitrile.
2.2 Concentration and cleanup: The extract is dried over sodium sulfate, concentrated using a
Kuderna-Danish evaporator, cleaned up (if necessary) using gel permeation chromatography
(GPC) and/or adsorption chromatography and/or solid-phase extraction, and then concentrated to
1 mL. Sulfur is removed from the extract, if required.
2.3 Gas chromatography: A 1-FL aliquot of the extract is injected into the gas chromatograph (GC).
The compounds are separated on a wide-bore, fused-silica capillary column. The analytes are
detected by an electron capture, microcoulometric, or electrolytic conductivity detector.
2.4 Identification of a pollutant (qualitative analysis) is performed by comparing the GC retention
times of the compound on two dissimilar columns with the respective retention times of an
authentic standard. Compound identity is confirmed when the retention times agree within their
respective windows.
2.5 Quantitative analysis is performed using an authentic standard to produce a calibration factor or
calibration curve, and using the calibration data to determine the concentration of a pollutant in
the extract. The concentration in the sample is calculated using the sample weight or volume and
the extract volume.
2.6 Quality is assured through reproducible calibration and testing of the extraction and GC systems.
3. CoNTAuIN. liON AND INTERFERENCES
3.1 Solvents, reagents, glassware, and other sample-processing hardware may yield artifacts and/or
elevated baselines causing misinterpretation of chromatograms. All materials used in the analysis
shall be demonstrated to be free from interferences under the conditions of analysis by running
method blanks as described in Section 8.5.
3.2 Glassware and, where possible, reagents are cleaned by solvent rinse and baking at 450°C for
1 hour minimum in a muffle furnace or kiln. Some thermally stable materials, such as PCBs, may
not be eliminated by this treatment, and thorough rinsing with acetone and pesticide-quality hexane
may be required.
3.3 Specific selection of reagents and purification of solvents by distillation in all-glass systems may
be required.
638
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Method 1656
3.4 Interference by phthalate esters can pose a major problem in pesticide analysis when using the
electron capture detector. Phthalates usually appear in the chromatogram as large, late-eluting
peaks. Phthalates may be leached from common flexible plastic tubing and other plastic materials
during the extraction and cleanup processes. Cross-contamination of clean glassware routinely
occurs when plastics are handled during extraction, especially when solvent-wetted surfaces are
handled. Interferences from phthalates can best be minimized by avoiding the use of plastics in
the laboratory, or by using a microcoulometric or electrolytic conductivity detector.
3.5 Interferences coextracted from samples will vary considerably from source to source, depending
on the diversity of the site being sampled. The cleanup procedures given in this method can be
used to overcome many of these interferences, but unique samples may require additional cleanup
to achieve the minimum levels given in Table 2.
4. SAFETY
4.1 The toxicity or carcinogenicity of each compound or reagent used in this method has not been
precisely determined; however, each chemical compound should be treated as a potential health
hazard. Exposure to these compounds should be reduced to the lowest possible level. The
laboratory is responsible for maintaining a current awareness file of OSHA regulations regarding
the safe handling of the chemicals specified in this method. A reference file of material handling
sheets should also be made available to all personnel involved in these analyses. Additional
information on laboratory safety can be found in References 1 through 3.
4.2 The following compounds covered by this method have been tentatively classified as known or
suspectedhuman or mammalian carcinogens: 4,4’-DDD, 4,4’-DDT, the BHCs and the PCBs.
Primary standards of these compounds shall be prepared in a hood, and a NIOSH/MESA-approved
toxic gas respirator should be worn when high concentrations are handled.
4.3 Mercury vapor is highly toxic. If mercury is used for sulfur removal, all operations involving
mercury shall be performed in a hood.
4.4 Unknown samples may contain high concentrations of volatile toxic compounds. Sample
containers should be opened in a hood and handled with gloves that will prevent exposure. The
oven used for sample drying to determine percent moisture should be located in a hood so that
vapors from samples do not create a health hazard in the laboratory.
5. APPARA TLJS AND MA TERIALS
NOTE: Brand names, suppliers, and part numbers are for illustrative purposes only. No
endorsement is implied. Equivalent performance may be achieved using apparatus and
materials other than those specified here, but demonstration of equivalent performance
meeting requirements of this method is the responsibility of the laboratory.
5.1 Sampling equipment for discrete or composite sampling.
5.1.1 Sample bottles and caps.
5.1 .1.1 Liquid samples (waters, sludges, and similar materials that contain less than
5% solids): Sample bottle, amber glass, 1-L or 1-quart, with screw-cap.
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Method 1656
5.1.1.2 Solid samples (soils, sediments, sludges, filter cake, compost, and similar
materials that contain >5% solids): Sample bottle, wide mouth, amber glass,
500-mL minimum.
5.1.1.3 If amber bottles are not available, samples shall be protected from light.
5.1.1.4 Bottle caps: Threaded to fit sample bottles. Caps shall be lined with PTFE.
5.1.1.5 Cleaning.
5.1.1.5.1 Bottles are detergent-water washed, then rinsed with solvent or
baked at 450°C for 1 hour minimum before use.
5.1.1.5.2 Liners are detergent-water washed, then rinsed with reagent water
and solvent, and baked at approximately 200°C for 1 hour
minimum prior to use.
5.1.2 Compositing equipment: Automatic or manual compositing system incorporating glass
containers cleaned per bottle-cleaning procedure above. Sample containers are kept at
0 to 4°C during sampling. Glass or PTFE tubing only shall be used. If the sampler uses
a peristaltic pump, a minimum length of compressible silicone rubber tubing may be used
in the pump only. Before use, the tubing shall be thoroughly rinsed with methanol,
followed by repeated rinsings with reagent water to minimize sample contamination. An
integrating flow meter is used to collect proportional composite samples.
5.2 Equipment for determining percent moisture.
5.21 Oven, capable of maintaining a temperature of 110°C (±5°C).
5.2.2 Desiccator.
5.2.3 Crucibles, porcelain.
5.2.4 Weighing pans, aluminum.
5.3 Extraction equipment.
5.3.1 Equipment for ultrasonic extraction.
5.3.1.1 Sonic disrupter: 375 watt with pulsing capability and ½- or M -inch disrupter
horn (Ultrasonics, mc, Model 375C, or equivalent).
5.31.2 Sonabox (or equivalent), for use with disrupter.
5.3.2 Equipment for liquid-liquid extraction.
5.3.2.1 Continuous liquid-liquid extractor: PTFE or glass connecting joints and
stopcocks without lubrication, 1.5- to 2-L capacity (Hershberg-Wolf
Extractor, Cal-Glass, Costa Mesa,California, 1000- or 2000-mL continuous
extractor, or equivalent).
53.2.2 Round-bottom flask, 500-mL, with heating mantle.
5.3.2.3 Condenser, Graham, to fit extractor.
5.3.2.4 pH meter, with combination glass electrode.
5.3.2.5 pH paper, wide range (Hydrion Papers, or equivalent).
5.2.3 Separatory funnels: 250-, 500-, 1000-, and 2000-mL, with PTFE stopcocks.
5.3.4 Filtration apparatus.
640
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Method 1656
5.3.4.1 Glass powder funnels: 125- to 250-mL.
5.3.4.2 Filter paper for above (Whitman 41, or equivalent).
5.3.5 Beakers.
5.3.5.1 1.5- to 2-L, calibrated to 1 L.
5.3.5.2 400- to 500-mL.
5.3.6 Spatulas: Stainless steel or PTFE.
5.3.7 Drying column: 400 mm long by 15 to 20 mm 11) Pyrex chromatographic column
equipped with coarse glass fit or glass wool plug.
5.3.7.1 Pyrex glass wool: Extracted with solvent or baked at 450°C for 1 hour
minimum.
5.4 Evaporation/concentration apparatus.
5.4.1 Kuderna-Danish (K-D) apparatus.
5.4.1.1 Evaporation flask: 500-mi. (Kontes K-570001-0500, or equivalent), attached
to concentrator tube with springs (Kontes K-662750-0012).
5.4.1.2 Concentrator tube: 10-mL, graduated (Kontes K-570050-1025, or equivalent)
with calibration verified. Ground-glass stopper (size 19/22 joint) is used to
prevent evaporation of extracts.
5.4.1.3 Snyder column: Three-ball macro (Kontes K-503000-0232, or equivalent).
5.4.1.4 Snyder column: Two-ball micro (Kontes K-469002-0219, or equivalent).
5.4.1.5 Boiling chips.
5.4.1.5.1 Glass or silicon carbide: Approximately 10/40 mesh, extracted
with methylene chloride and baked at 450°C for 1 hour
minimum.
5.4.1.5.2 PTFE (optional): Extracted with methylene chloride.
5.4.2 Water bath: Heated, with concentric ring cover, capable of temperature control (±2°C),
installed in a fume hood.
5.4.3 Nitrogen evaporation device: Equipped with heated bath that can be maintained at 35 to
4.0°C (N-Evap, Organomation Associates, Inc., or equivalent).
5.4.4 Sample vials: Amber glass, 1- to 5-mL with PTFE-lined screw- or crimp-cap, to fit GC
autosampler.
5.5 Balances.
5.5.1 Analytical: Capable of weighing 0.1 mg.
5.5.2 Top loading: Capable of weighing 10 mg.
5.6 Apparatus for sample cleanup.
5.6.1 Automated gel permeation chromatograph (Analytical Biochemical Labs, Inc., Columbia,
MO, Model GPC Autoprep 1002, or equivalent).
5.6.1.1 Column: 600 to 700 mm long by 25 mm ID, packed with 70 g of SX-3
Bio-beads (Bio-Rad Laboratories, Richmond, CA, or equivalent).
641
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Method 1656
5.6.1.2 Syringe, 1O-mL, with Luer fitting.
5.6.1.3 Syringe-filter holder, stainless steel, and glass fiber or PTFE filters (Gelman
Acrodisc-CR, 1 to 5 micron, or equivalent).
5.6.1.4 UV detector: 254-nm, preparative or semi-prep flow cell: (Isco, Inc., Type
6; Schmadzu, 5 mm path length; Beckman-Altex 152W, 8 pL micro-prep
flow cell, 2 mm path; Pharmacia UV-1, 3 mm flow cell; LDC Milton-Roy
UV-3, monitor #1203; or equivalent).
5.6.2 Vacuum system and cartridges for solid-phase extraction (SPE).
5.6.2.1 Vacuum system: Capable of achieving 0.1 bar (house vacuum, vacuum
pump, or water aspirator), with vacuum gauge.
5.6.2.2 VacElute Manifold (Analytichem International, or equivalent).
5.6.2.3 Vacuum trap: Made from 500-mL sidearm flask fitted with single-hole
rubber stopper and glass tubing.
5.6.2.4 Rack for holding 50-mL volumetric flasks in the manifold.
5.6.2.5 Column: Mega Bond Elut, Non-polar, C18 Octadecyl, 10 g/60 mL
(Analytichem International Cat. No. 607H060, or equivalent).
5.6.3 Chromatographic column: 400 mm long by 22 mm ID, with PTFE stopcock and coarse
fit (Kontes K-42054, or equivalent).
5.6.4 Sulfur removal tubes: 40- to 50-mL bottle or test tube with PTFE-lined screw-cap.
5.7 Centrifuge apparatus.
5.7.1 Centrifuge: Capable of rotating 500-mL centrifuge bottles or 15-mL centrifuge tubes at
5,000 rpm minimum.
5.72 Centrifuge bottles: 500-mL, with screw-caps, to fit centrifuge.
5.7.3 Centrifuge tubes: 12- to 15-mL, with screw-caps, to fit centrifuge.
5.7.3 Funnel, Buchner, 15 cm.
5.7.3.1 Flask, filter, for use with Buchner funnel.
5.7.3.2 Filter paper, 15 cm (Whatman #41, or equivalent).
5.8 MIscellaneous glassware.
5.8.1 Pipettes, glass, volumetric, 1.00-, 5.00-, and 10.0-mL.
5.8.2 Syringes, glass, with Luerlok tip, 0.1-, 1.0- and 5.0-mL. Needles for syringes, 2-inch,
22-gauge.
5.8.3 Volumetric flasks, 10.0-, 25.0-, and 50.0-mL.
5.8.4 Scintillation vials, glass, 20- to 50-mL, with PTFE-lined screw-caps.
5.9 Gas chromatograph: Shall have splitless or on-column simultaneous automated injection into
separate capillary columns with a halide-specific detector at the end of each column, temperature
program with isothermal holds, data system capable of recording simultaneous signals from the
two detectors, and shall meet all of the performance specifications in Section 14.
5.9.1 GC columns: Bonded-phase, fused-silica capillary.
642
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Method 1656
5.9.1.1 Primary: 30 m (±3 m) long by 0.5 mm (±0.05 mm) ID DB-608 (or
equivalent).
5.9.1.2 Confirmatory: DB-l701, or equivalent, with same dimensions as primary
column.
5.9.2 Data system: Shall collect and record GC data, store GC runs on magnetic disk or tape,
process GC data, compute peak areas, store calibration data including retention times and
calibration factors, identify OC peaks through retention times, compute concentrations,
and generate reports.
5.9.2.1 Data acquisition: GC data shall be collected continuously throughout the
analysis and stored on a mass storage device.
5.9.2.2 Calibration factors and calibration curves: The data system shall be used to
record and maintain lists of calibration factors, and multi-point calibration
curves (Section 7). Computations of relative standard deviation (coefficient
of variation) are used for testing calibration linearity. Statistics on initial
(Section 8.2) and ongoing (Section 14.6) performance shall be computed and
maintained.
5.9.2.3 Data processing: The data system shall be used to search, locate, identify,
and quantify the compounds of interest in each GC analysis. Software
routines shall be employed to compute and record retention times and peak
areas. Displays of chromatograins and library comparisons are required to
verify results.
5.9.3 Halide-specific detector: Electron capture or electrolytic conductivity (Micoulometric,
Hall, 0.!., or equivalent), capable of detecting 8 pg of aidrin under the analysis
conditions given in Table 2.
6. REAGENTS AND STANDARDS
6.1 Sample preservation: Sodium thiosulfate (ACS), granular.
6.2 pH adjustment.
6.2.1 Sodium hydroxide: Reagent grade.
6.2.1.1 Concentrated solution (iON): Dissolve 40 g NaOH in 100 mL reagent water.
6.2.1.2 Dilute solution (0.1M): Dissolve 4 g NaOH in 1 L of reagent water.
6.2.2 Sulfuric acid (1 + 1): Reagent grade, 6N in reagent water. Slowly add 50 mL H 2 S0 4
(specific gravity 1.84) to 50 mL reagent water.
6.2.3 Potassium hydroxide: 37% (W/V). Dissolve 37 g KOH in 100 mL reagent water.
6.3 Solution drying and back-extraction.
6.3.1 Sodium sulfate, reagent grade, granular anhydrous (Baker 3375, or equivalent), rinsed
with methylene chloride (20 rnLlg), baked at 450°C for 1 hour minimum, cooled in a
desiccator, and stored in a pre-cleaned glass bottle with screw-cap which prevents
moisture from entering.
643
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Method 1656
6.3.2 Sodium sulfate solution: 2% (W/V) in reagent water, pH-adjusted to 8.5 to 9.0 with
KOH or H 2 S0 4 .
6.4 Solvents: Methylene chloride, hexane, ethyl ether, acetone, acetonitrile, isooctane, and methanol;
pesticide-quality; lot-certified to be free of interferences.
6.4.1 Ethyl ether must be shown to be free of peroxides before it is used, as indicated by EM
Laboratories Quant Test Strips (Scientific Products P1126-8, or equivalent). Procedures
recommended fur removal of peroxides are provided with the test strips. After cleanup,
20 mL of ethyl alcohol is added to each liter of ether as a preservative.
6.5 QPC calibration solution: Solution containing 300 mg/mL corn oil, 15 mg/mL bis (2-ethyihexyl)
phthalate, 1.4 rng/mL pentachlorophenol, 0.1 mg/mL perylene, and 0.5 mglmL sulfur.
6.6 Sample cleanup.
6.6.1 Florisil: PR grade, 60/100 mesh, activated at 650 to 700°C, stored in the dark in glass
container with PTFE-lined screw-cap. Activate at 130°C for 16 hours minimum
Immediately prior to use. Alternatively, 500-mg cartridges (J.T. Baker, or equivalent)
may be used.
6.6.2 Solid-phase extraction.
6.6.2.1 SPE cartridge calibration solution: 2,4,6-trichlorophenol, 0.1 ug/mL in
acetone.
6.62.2 SPE elution solvent: Methylene chloride:acetonitrile:hexane (50:3:47).
6.6.3 Alumina, neutral, Brockman Activity I, 80 to 200 mesh (Fisher Scientific Certified, or
equivalent). Heat for 16 hours at 400 to 450°C. Seal and cool to room temperature.
Add 7% (W/W) reagent water and mix for 10 to 12 hours. Keep bottle tightly sealed.
6.6.4 Siicic acid, 100 mesh.
6.6.5 Sulfur removal: Mercury (triple-distilled), copper powder (bright, non-oxidized), or
TBA sodium sulfite. if mercury is used, observe the handling precautions in Section 4.
6.7 Reagent water: Water in which the compounds of interest and interfering compounds are not
detected by this method.
6.8 High-solids reference matrix: Playground sand or similar material in which the compounds of
interest and interfering compounds are not detected by this method. May be prepared by
extraction with methylene chloride and/or baking at 450°C for 4 hours minimum.
6.9 Standard solutions: Purchased as solutions or mixtures with certification to their purity,
concentration, and authenticity, or prepared from materials of known purity and composition. If
compound purity is 96% or greater, the weight may be used without correction to compute the
concentration of the standard. When not being used, standards are stored in the dark at -20 to
-10°C in screw-capped vials with PTFE-lined lids. A mark is placed on the vial at the level of
the solution so that solvent evaporation loss can be detected. The vials are brought to room
temperature prior to use. Any precipitate is redissolved and solvent is added if solvent loss has
occurred.
6.10 Preparation of stock solutions: Prepare in isooctane per the steps below. Observe the safety
precautions in Section 4.
644
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Method 1656
6.10.1 Dissolve an appropriate amount of assayed reference material in solvent. For example,
weigh 10 mg aidrin in a 10-mL ground-glass stoppered volumetric flask and fill to the
mark with isooctane. After the aidrin is completely dissolved, transfer the solution to a
15-mL vial with PTFE-lined cap.
6.10.2 Stock solutions should be checked for signs of degradation prior to the preparation of
calibration or performance test standards.
6.10.3 Stock solutions shall be replaced after 6 months, or sooner if comparison with quality
control check standards indicates a change in concentration.
6.11 Secondary mixtures: Using stock solutions (Section 6.10), prepare mixtures at the levels shown
in Table 3 for calibration and calibration verification (Sections 7.3 and 14.5), for initial and
ongoing precision and recovery (Sections 8.2 and 14.6), and for spiking into the sample matrix
(Section 8.4).
6.12 Surrogate spiking solution: Prepare dibutyl chlorendate (OBC) at aconcentration of 2 ng/mL in
acetone.
NOTE: If DBC is nor available, compounds such as tetrachloro-m-xylene or
decachiorobiphenyl may be used provided that the laboratory peiforms the tests described
in Section 8.2 using these compounds.
6.13 DDT and endrin decomposition solution: Prepare a solution containing endrin at a concentration
of 1 pg/mL and DDT at a concentration of 2 p g/mL.
6.14 Stability of solutions: All standard solutions (Sections 6.9 through 6.13) shall be analyzed within
48 hours of preparation and on a monthly basis thereafter for signs of degradation. Standards will
remain acceptable if the peak area remains within ± 15% of the area obtained in the initial analysis
of the standard.
7. SETUP AND CAL/BRA 770N
7.1 Configure the GC system as given in Section 5.9 and establish the operating conditions in Table 2.
7.2 Attainment of method detection limit (MDL) and DDTIEndrin decomposition requirements:
Determine that each column/detector system meets the MDLs (Table 2), and the DDT and Endrin
decomposition test (Section 13.4).
7.3 Calibration.
7.3.1 Injection of calibration solutions.
7.3.1.1 Compounds with calibration data in Table 3: The compounds in each
calibration group in Table 3 were chosen so that each compound would be
separated from the others by approximately 1 minute on the primary column.
The concentrations were chosen to bracket the working range of either the
ECD or the ELCD. However, because the response of the ECLD is less for
some compounds than that of the ECD, it may be necessary to inject a larger
volume of calibration solution when the ELCD is used.
645
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Method 1656
7.3.1.2 Compounds without calibration data in Table 3: Prepare calibration standards
at a minimum of three concentration levels. One of these concentrations
should be near, but above, the MDL (Fable 2) and the other concentrations
should define the working range of the detectors.
7.3.1.3 Set the automatic injector to inject a constant volume in the range of 0.5 to
5.0 1 tL of each calibration solution into the GC column/detector pairs,
beginning with the lowest level mixture and proceeding to the highest. For
each compound, compute and store, as a function of the concentration
injected, the retention time and peak area on each column/detector system
(primary and confirmatory). For the multi-componenent analytes (PCBs,
toxaphene), store the retention time and peak area for the five largest peaks.
7.3.2 Retention time: The polar nature of some analytes causes the retention time to decrease
as the quantity injected increases. To compensate this effect, the retention time for
compound identification is correlated with the analyte level.
7.3.2.1 If the difference between the maximum and minimum retention times for any
compound is less than 5 seconds over the calibration range, the retention time
for that compound can be considered constant and an average retention time
may be used for compound identification.
7.3.2.2 Retention time calibration curve (retention time vs. amount): If the retention
time for a compound in the lowest level standard is more than 5 seconds
greater than the retention time for the compound in the highest level standard,
a retention time calIbration curve shall be used for identification of that
7.3.3 Calibration factor (ratio of area to amount injected).
7.3.3.1 Compute the coefficient of variation (relative standard deviation) of the
calibration factor over the calibration range for each compound on each
column/detector system.
7.3.3.2 Linearity: If the calibration factor for any compound is constant (C <20%)
over the calibration range, an average calibration factor may be used for that
compound; otherwise, the complete calibration curve (area vs. amount) for
that compound shall be used.
7.4 Combined QC standards: To preclude periodic analysis of all of the individual calibration groups
of compounds (Table 3), the GC systems are calibrated with combined solutions as a final step.
NotallofthecompoundsinthesestandardswilbeseparatedbytheGCcolumnsusedinthis
method. Retention times and calibration factors are verified for the compounds that are resolved,
and calibration factors are obtained for the unresolved peaks.
7.4.1 Analyze the combined QC standard on each column/detector pair.
7.4,1.1 For those compounds that exhibit a single, resolved GC peak, the retention
time shall be within ±5 seconds of the retention time of the peak in the
medium level calibration standard (Table 3), and the calibration factor using
the primary column shall be within ±20% of the calibration factor in the
medium level standard (Table 3).
646
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Method 1656
7.4.1.2 For the peaks containing two or more compounds, compute and store the
retention times at the peak maxima on both columns rimary and
confirmatory), and also compute and store the calibration factors on both
columns. These results will be used for calibration verification (Section 13.2
and 13.5) and for precision and recovery studies (Sections 8.2 and 13.6).
7.5 Florisil calibration: The cleanup procedure in Section 11 utilizes Florisil column chromatography.
Florisil from different batches or sources may vary in adsorptive capacity. To standardize the
amount of Florisil that is used, the use of the lauric acid value (Reference 4) is suggested. The
referenced procedure determines the adsorption of lauric acid (in milligrams per gram of Florisil)
from hexane solution. The amount of Florisil to be used for each column is calculated by dividing
110 by this ratio and multiplying by 20 g.
8. QuALITY CONTROL
8.1 Each laboratory that uses this method is required to operate a formal quality control program
(Reference 5). The minimum requirements of this program consist of an initial demonstration of
laboratory capability, an ongoing analysis of standards and blanks as tests of continued
performance, and analysis of spiked samples to assess accuracy. Laboratory performance is
compared to established performance criteria to determine if the results of analyses meet the
performance characteristics of the method. If the method is to be applied routinely to samples
containing high solids with very little moisture (e.g., soils, compost), the high-solids reference
matrix (Section 6.8) is substituted for reagent water (Section 6.7) in all performance tests, and the
high-solids method (Section 10) is used for these tests.
8.1.1 The analyst shall make an initial demonstration of the ability to generate acceptable
accuracy and precision with this method. This ability is established as described in
Section 8.2.
8.1.2 The analyst is permitted to modify this method to improve separations or lower the costs
of measurements, provided all performance requirements are met. Each time a
modification is made to the method or a cleanup procedure is added, the analyst is
required to repeat the procedure in Section 8.2 to demonstrate method performance. If
detection limits will be affected by the modification, the analyst is required to repeat the
demonstration of detection limits (Section 7.2).
8.1.3 The laboratory shall spike all samples with at least one surrogate compound to monitor
method performance. This test is described in Section 8.3. When results of these spikes
indicate atypical method performance for samples, the samples are diluted to bring
method performance within acceptable limits (Section 16).
8.1.4 The laboratory shall, on an ongoing basis, demonstrate through calibration verification
and the analysis of the combined QC standard (Section 6.11) that the analysis system is
in control. These procedures are described in Sections 13.1, 13.5, and 13.6.
8.1.5 The laboratory shall maintain records to define the quality of data that is generated.
Development of accuracy statements is described in Section 8.4.
8.1.6 Analyses of blanks are required to demonstrate freedom from contamination. The
procedures and criteria for analysis of a blank are described in Section 8.5.
647
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Method 1656
8.1.7 Other analytes may be determined by this method. The procedure for establishing a
preliminary quality control linilt for a new analyte is given in Section 8.6.
8.2 Initial precision and recovery: To establish the ability to generate acceptable precision and
accuracy, the analyst shall perform the following operations.
0.2.1 For analysis of samples containing low solids (aqueous samples), extract, concentrate,
and analyze one set of four l-L aliquots of reagent water spiked with the combined QC
standard (Section 6.11) according to the procedure in Section 10. Alternatively, sets of
four replicates of the individual calibration groups (Fable 3) may be used. For samples
containing high solids, a set of four 30-g aliquots of the high-solids reference matrix are
used.
8.2.2 Using results of the set of four analyses, compute the average percent recovery (X) and
the coefficient of variation (Ci,) of percent recovery (s) for each compound.
8.2.3 For each compound, compare s and X with the corresponding limits for initial precision
and accuracy in Table 4. For coeluting compounds, use the coeluted compound with the
least restrictive specification (largest C. and widest range). If s and X for all compounds
meet the acceptance criteria, system performance is acceptable and analysis of blanks and
samples may begin. If, however, any individual s exceeds the precision limitor any
individual X falls outside the range for accuracy, system performance is unacceptable for
that compound. In this case, correct the problem and repeat the test.
8.3 The laboratory shall spike all samples with at least one surrogate compound to assess method
performance on the sample matrix.
8.3.1 Analyze each sample according to the method beginning in Section 10.
8.3.2 Compute the percent recovery (P) of the DBC or other surrogate.
8.3.3 The surrogate recovery shall be 40 to 120%. If the recovery of the surrogate falls
outside of these limits, method performance is unacceptable for that sample, and the
sample is complex. Water samples are diluted, and smaller amounts of soils, sludges,
and sediments are reanalyzed per Section 16.
8.4 Method accuracy: The laboratory shall spike (matrix spike) at least 10% of the samples from a
given site type (e.g., influent to treatment, treated effluent, produced water, river sediment). If
only one sample from a given site type is analyzed, a separate aliquot of that sample shall be
spiked.
8.4.1 The concentration of the matrix spike shall be determined as follows:
8.4.1.1 If, as in compliance monitoring, the concentration of a specific analyte in the
sample is being checked against a regulatory concentration limit, the matrix
spike shall be at that limit or at 1 to 5 times higher than the background
concentration determined in Section 8.4.2, whichever concentration is larger.
8.4.1.2 If the concentration of an analyte in the sample is not being checked against
a limit specific to that analyte, the matrix spike shall be at the concentration
of the combined QCstandard (Fable 3)or at ito 5 times higher than the
background concentration, whichever concentration is larger.
8.4.1.3 If it is impractical to determine the background concentration before spiking
(e.g., maximum holding times will be exceeded), the matrix spike
648
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Method 1656
concentration shall be the regulatory concentration limit, if any; otherwise,
the larger of either 5 times the expected background concentration or at the
concentration of the combined QC standard (Table 3).
8.4.2 Analyze one sample aliquot to determine the background concentration (B) of each
analyte. If necessary, prepare a standard solution appropriate to produce a level in the
sample 1 to 5 times the background concentration. Spike a second sample aliquot with
the standard solution and analyze it to determine the concentration after spiking (A) of
each analyte. Calculate the percent recovery (P) of each analyte:
Equation 1
= 100(A-B )
T
where
T = True value of the spike
8.4.3 Compare the percent recovery for each analyte with the corresponding QC acceptance
criteria in Table 4. If any analyte fails the acceptance criteria for recovery, the sample
is complex and must be diluted and reanalyzed per Section 16.
8.4.4 As part of the QC program for the laboratory, method accuracy for samples shall be
assessed and records shall be maintained. After the analysis of five spiked samples of
a given matrix type (water, soil, sludge, sediment) in which the analytes pass the tests
in Section 8.4.3, compute the average percent recovery (P) and the standard deviation of
the percent recovery (sr) for each compound (or coeluting compound group). Express
the accuracy assessment as a percent recovery interval from P - 2s to P + 2 s for each
matrix. For example, if P=90% and s = 10% for five analyses of compost, the accuracy
interval is expressed as 70 to 110%. Update the accuracy assessment for each compound
in each matrix on a regular basis (e.g., after each five to ten new accuracy
measurements).
8.5 Blanks: Reagent water and high-solids reference matrix blanks are analyzed to demonstrate
freedom from contamination.
8.5.1 Extract and concentrate a 1-L reagent water blank or a 30-g high-solids reference matrix
blank with each sample batch (samples started through the extraction process on the same
8-hour shift, to a maximum of 20 samples). Analyze the blank immediately after analysis
of the combined QC standard (Section 13.6) to demonstrate freedom from contamination.
8.5.2 If any of the compounds of interest (Fable 1) or any potentially interfering compound is
found in an aqueous blank at greater than 0.05 g/L, or in a high-solids reference matrix
blank at greater than 1 pg/kg (assuming the same calibration factor as aidrin for
compounds not listed in Table 1), analysis of samples is halted until the source of
contamination is eliminated and a blank shows no evidence of contamination at this level.
8.6 Other analytes may be determined by this method. To establish a quality control limit for an
analyte, determine the precision and accuracy by analyzing four replicates of the analyte along
with the combined QC standard per the procedure in Section 8.2. If the analyte coelutes with an
649
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Method 1656
analyte in the QC standard, prepare a new QC standard without the coeluting component(s).
Compute the average percent recovery (A) and the standard deviation of percent recovery (sn) for
the analyte, and measure the recovery and standard deviation of recovery for the other analytes.
The data for the new analyte is assumed to be valid if the precision and recovery specifications
for the other analytes are met otherwise, the analytical problem is corrected and the test is
repeated. Establish a preliminary quality control limit of A ± 2s., for the new analyte and add
the limit to Table 4.
8.7 The specifications contained in this method can be met if the apparatus used is calibrated properly,
then maintained in a calibrated state. The standards used for calibration (Section 7), calibration
verification (Section 13.5), and for initial (Section 8.2) and ongoing (Section 13.6) precision and
recovery should be identical, so that the most precise results will be obtained. The GC
instruments will provide the most reproducible results if dedicated to the settings and conditions
required for the analyses of the analytes given n this method.
8.8 Depending on specific program requirements, field replicates and field spikes of the analytes of
interest into samples may be required to assess the precision and accuracy of the sampling and
sample transporting techniques.
9. SA*q’LE COLLECTiON, PRESERVATION, AND HANDLING
9.1 Collect samples in glass containers following conventional sampling practices (Reference 6),
except that the bottle shall not be prerinsed with sample before collection. Aqueous samples
which flow freely are collected in refrigerated bottles using automatic sampling equipment. Solid
samples are collected as grab samples using wide-mouth jars.
9.2 Mahdain samples at 0 to 4°C from the time of collection until extraction. If the samples will not
be extracted within 72 hours of collection, adjust the sample to a pH of 5.0 to 9.0 using sodium
hydn xide or sulfuric acid solution. Record the volume of acid or base used. If residual chlorine
is present in aqueous samples, add 80 mg sodium thiosulfate per liter of water. EPA Methods
330.4 and 330.5 may be used to measure residual chlorine (Reference 7).
9.3 Begin sample extraction within 7 days of collection, and analyze all extracts within 40 days of
extraction.
10. SAMPLE EXTRACTiON AND CONCENTRA liON
Samples col aining 1% solids or less are extracted directly using continuous liquid-liquid extraction
techniques (Section 10.2.1). Samples containing 1 to 30% solids are diluted to the 1% level with reagent
water and extracted using continuous liquid-liquid extraction techniques (Section 10.2.2). Samples
containing mere than 30% solids are extracted using ultrasonic techniques (Section 10.2.5). Figure 1
outlines the extraction and concentration steps.
10.1 Detenninationofpercentsolids.
10.1.1 Weigh 5 to lOg of sample into a tared beaker. Record the weight to three significant
figures.
10.1.2 Dry overnight (12 hours minimum) at 110°C (±5°C), and cool in a dessicator.
10.1.3 Determine percent solids as follows:
650
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Method 1656
Equation 2
% soiicis = weight of dry sample ioo
weight of wet sample
10.2
Preparation of samples for extraction
10.2.1 Samples containing 1% solids or less: Extract the sample directly using continuous
liquid-liquid extraction techniques.
10.2.1.1 Measure 1.00 L (±0.01 L) of sample into a clean 1.5- to 2.0-L beaker.
10.2.1.2 Spike 0.5 mL of the surrogate spiking solution (Section 6.12) into the sample
aliquot. Proceed to preparation of the QC aliquots for low solids samples
(Section 10.2.3).
10.2.2 Samples containing 1 to 30% solids.
10.2.2.1 Mix sample thoroughly.
10.2.2.2 Using the percent solids found in Section 10.1.3, determine the weight of
sample required to produce 1 L of solution containing 1% solids as follows:
Equation 3
l000g
sample weight = % solids
10.2.2.3 Place the weight determined in Section 10.2.2.2 in a clean 1.5- to 2.0-L
beaker. Discard all sticks, rocks, leaves, and other foreign material prior to
weighing.
10.2.2.4 Bring the volume of the sample aliquot(s) to 100 to 200 mL with reagent
water.
10.2.2.5 Spike 0.5 mL of the surrogate spiking solution (Section 6.12) into each
sample aliquot.
10.2.2 .6 Using a clean metal spatula, break any solid portions of the sample into small
pieces.
10.2.2.7 Place the %-inch horn on the ultrasonic probe approximately ½ inch below
the surface of each sample aliquot and pulse at 50% for 3 minutes at full
power. If necessary, remove the probe from the solution and break any large
pieces using the metal spatula or a stirring rod and repeat the sonication.
Clean the probe with methylene chloride:acetone (1:1) between samples to
preclude cross-contamination.
10.2.2.8 Bring the sample volume to 1.0 L (±0.1 L) with reagent water.
651
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Method 1656
10.2.3 Preparation of QC aliquots for samples containing low solids (less than 30%).
10.2.3.1 For each sample or sample batch (to a maximum of 20) to be extracted at the
same time, place two 1.0 L (±0.01 L) aliquots of reagent water in clean 1.5-
to 2.0-L beakers.
10.2.3.2 Blank: Spike 0.5 mL of the surrogate spiking solution (Section 6.12) into one
reagent water aliquot.
10.2.3.3 Spike the combined QC standard (Section 7.4) into the remaining reagent
water aliquot.
10.2.3.4 If a matrix spike is required, prepare an aliquot at the concentrations specified
in Section 8.4.
10.2.4 Stir and equilibrate all sample and QC solutions for 1 to 2 hours. Extract the samples
and QC aliquots per Section 10.3.
10.2.5 Samples containing 30% solids or more.
10.2.5.1 Mix the sample thoroughly.
10.2.5.2 Weigh 30 g (±0.3 g) into a clean 400- to 500-mL beaker. Discard all sticks,
rocks, leaves, and other foreign material prior to weighing.
10.2.5.3 Spike 0.5 mL of the surrogate spiking solution (Section 6.12) into the aliquot.
10.2.5.4 QC aliquot: For each sample or sample batch (to a maximum of 20) to be
extracted at the same time, place 30 g (± 0.3 g) of the high-solids reference
matrix in each of two clean 400- to 500-mL beakers.
10.2.5.5 Blank: Spike 0.5 mL of the surrogate spiking solution (Section 6.12) into one
aliquot of the high-solids reference matrix.
10.2.5.6 Spike the combined QC standard (Section 6.11) into the remaining high-
solids reference matrix aliquot. Extract the high-solids samples per
Section 10.4.
10.3 Continuous extraction of low-solids (aqueous) samples: Place 100 to 150 mL methylene chloride
ineachcontinuousextractorand200to300mLineach distilhingflask.
10.3.1 Pour the sample(s), blank, and standard aliquots into the extractors. Rinse the glass
containers with 50 to 100 mL methylene chloride and add to the respective extractors.
Include all solids in the extraction process.
10.3.2 Extraction: Adjust the pH of the waters in the extractors to 5 to 9 with NaOH or H 2 S0 4
while monitoring with a pH meter. Caution: Some samples require acidification in a
hood because of the potential for generating hydrogen sulfide.
103.3 Begin the extraction by heating the flask until the methylene chloride is boiling When
properly adjusted, one to two drops of methylene chloride per second will fall from the
condensertipintothewater. Testandadjustthepllofthewatersduringthefirstl to
2 hours of extraction. Extract for 18 to 24 hours.
10.3.4 Remove the distilling flask, estimate and record the volume of extract (to the nearest
100 mL), and pour the contents through a prerinsed drying column containing 7 to 10
cm of anhydrous sodium sulfate. Rinse the distilling flask with 30 to 50 mL of
methylene chloride and pour through the drying column. For extracts to be cleaned up
652
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Method 1656
using GPC, collect the solution in a 500-mi. K-D evaporator flask equipped with a
10-mL concentrator tube. Seal, label the pesticide and herbicide fractions, and
concentrate per Sections 10.5 to 10.6.
10.4 Ultrasonic extraction of high solids samples: Procedures are provided for extraction of non-
municipal sludge (Section 10.4.1) and municipal sludge samples (Section 10.4.2).
10.4.1 Ultrasonic extraction of non-municipal sludge high-solids aliquots.
10.4.1.1 Add 60 to 70 g of powdered sodium sulfate to the sample and QC aliquots.
Mix each aliquot thoroughly. Some wet sludge samples may require more
than 70 g for complete removal of water. All water must be removed prior
to addition of organic solvent so that the extraction process is efficient.
10.4.1.2 Add 100 mL (± 10 mL) of acetone:methylene chloride (1:1) to each of the
aliquots and mix thoroughly.
10.4.1.3 Place the M-inch horn on the ultrasonic probe approximately ‘Ii inch below
the surface of the solvent but above the solids layer and pulse at 50% for
3 minutes at full power. If necessary, remove the probe from the solution
and break any large pieces using a metal spatula or a stirring rod and repeat
the sonication.
10.4.1.4 Decant the extract through a prerinsed drying column containing 7 to 10 cm
anhydrous sodium sulfate into a 500- to 1000-mL graduated cylinders.
10.4.1.5 Repeat the extraction steps (Sections 10.4.1.3 to 10.4.1.4) twice more for
each sample and QC aliquot. On the final extraction, swirl the sample or QC
aliquot, pour into its respective drying column, and rinse with
acetone:methylene chloride. Record the total extract volume. If necessary,
transfer the extract to a centrifuge tube and centrifuge for 10 minutes to settle
fine particles.
10.4.2 Ultrasonic extraction of high-solids municipal sludge aliquots.
10.4.2.1 Add 100 mL (± 10 mL) of acetonitrile to each of the aliquots and mix
thoroughly.
10.4.2.2 Place the M-inch horn on the ultrasonic probe approximately 1 h inch below
the surface of the solvent but above the solids layer and pulse at 50% for
3 minutes at full power. If necessary, remove the probe from the solution
and break any large pieces using a metal spatula or a stirring rod and repeat
the sonication.
10.4.2.3 Decant the extract through filter paper into a 1000- to 2000-mL separatory
funnel.
10.4.2.4 Repeat the extraction and filtration steps (Sections 10.4.2.2 to 10.4.2.3) using
a second 100 mL (± 10 niL) of acetonitrile.
10.4.2.5 Repeat the extraction step (Section 10.4.2.3) using 100 mL (±10 mL) of
methylene chloride. On this final extraction, swirl the sample or QC aliquot,
pour into its respective filter paper, and rinse with methylene chloride.
Record the total extract volume.
653
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Method 1656
10.4.2.6 For each extract, prepare 1.5 to 2 L of reagent water containing 2% sodium
sulfate. Adjust the pH of the water to 6.0 to 9.0 with NaOH or H 2 S0 4 .
10.4.2.7 Back-extract each extract three times sequentially with 500 mL of the aqueous
sodium sulfate solution, returning the bottom (organic) layer to the separatory
funnel the first two times while discarding the top (aqueous) layer. On the
final back extraction, filter each pesticide extract through a prerinsed drying
column cont2ining 7 to 10 cm anhydrous sodium sulfate into a 500- to
1000-niL graduated cylinder. Record the final extract volume.
10.4.3 For extracts to be cleaned up using GPC, filter these extracts through Whatman #41
paper into a 500-mL lCD evaporator flask equipped with a 10-mL concentrator tube.
Rinse the graduated cylinder or centrifuge tube with 30 to 50 mL of methylene chloride
and pour through filter to complete the transfer. Seal and label the K-D flasks.
Concentrate these fractions per Sections 10.5 through 10.8.
105 Macro concentration.
10.5.1 Concentrate the extracts in separate 500-mL K-D flasks equipped with 10-mL
concentrator tubes. Add one to two clean boiling chips to the flask and attach a
three-ball macro Snyder column. Prewet the column by adding approximately I mL of
methylene chloride through the top. Place the K-D apparatus in a hot water bath so that
the entire lower rounded surface of the flask is bathed with steam. Adjust the vertical
position of the apparatus and the water temperature as required to complete the
concentration in 15 to 20 minutes. At the proper rate of distillation, the balls of the
column will actively chatter but the chambers will not flood
10.5.2 When the liquid has reached an apparent volume of 1 mL, remove the K-D apparatus
front the bath and allow the solvent to drain and cool for at least 10 minutes.
105.3 If the extract is to be cleaned up using (3PC, remove the Snyder column and rinse the
flask and its lower joint into the concentrator tube with 1 to 2 mL of methylene chloride.
A 5-mL syringe is recommended for this operation. Adjust the final volume to 10 mL
and proceed to GPC cleanup in Section 11.
10.6 Hexane exchange: Extracts to be subjected to Florisil or silica gel cleanup and extracts that have
been cleaned up are exchanged into hexane
10.6.1 Remove the Snyder column, add approximately 50 mL of hexane and a clean boiling
chip, and reattach the Snyder column. Concentrate the extract as in Section 10.5 except
use hexane to prewet the column. The elapsed time of the concentration should be 5 to
10. minutes.
10.6.2 Remove the Snyder column and rinse the flask and its lower joint into the concentrator
tube with! 1o2 mLofhexane. Adjust the final volume of extracts that have not been
cleaned up by GPC to 10 mL and those that have been cleaned up by GPC to 5 mL (the
difference accounts for the 50% loss in the GPC cleanup). Clean up the extracts using
the Florisil, silica gel, and/or sulfur removal procedures in Section 11
654
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Method 1656
11. CLEANUP AND SEPARATION
11.1 Cleanup procedures may not be necessary for relatively clean samples (treated effluents, ground
water, drinking water). If particular circumstances require the use of a cleanup procedure, the
analyst may use any or all of the procedures below or any other appropriate procedure. However,
the analyst shall first repeat the tests in Section 8.2 to demonstrate that the requirements of Section
8.2 can be met using the cleanup procedure(s) as an integral part of the method. Figure 1
outlines the cleanup steps.
11.1.1 Gel permeation chromatography (Section 11.2) removes many high molecular weight
interferents that cause GC column performance to degrade. It is used for all soil and
sediment extracts and may be used for water extracts that are expected to contain high
molecular weight organic compounds (e.g., polymeric materials, humic acids).
11.1.2 The solid-phase extraction cartridge (Section 11.3) removes polar organic compounds
such as phenols.
11.1.3 The Florisil column (Section 11.4) allows for selected fractionation of the organo-chlorine
compounds and will also eliminate polar interferences.
11.1.4 Alumina column cleanup (Section 11.5) may also be used for cleanup of the
organo-chlorine compounds.
11.1.5 Elemental sulfur, which interferes with the electron capture gas chromatography of some
of the pesticides, is removed using GPC, mercury, or activated copper. Sulfur removal
(Section 11.6) is required when sulfur is known or suspected to be present.
11.2 Gel permeation chromatography (GPC).
11.2.1 Column packing.
11.2.1.1 Place 70 to 75 g of SX-3 Bio-beads in a 400- to 500-mL beaker.
11.2.1.2 Cover the beads with methylene chloride and allow to swell overnight (12
hours minimum).
11.2.1.3 Transfer the swelled beads to the column and pump solvent through the
column, from bottom to top, at 4.5 to 5.5 mL/min prior to connecting the
column to the detector.
11.2.1.4 After purging the column with solvent for 1 to 2 hours, adjust the column
head pressure to 7 to 10 psig, and purge for 4 to 5 hours to remove air.
Maintain a head pressure of 7 to 10 psig. Connect the column to the
detector.
11.2.2 Column calibration.
11.2.2.1 Load 5 mL of the calibration solution (Section 6.5) into the sample loop.
11.2.2.2 Inject the calibration solution and record the signal from the detector. The
elution pattern will be corn oil, bis (2-ethythexyl) phthalate,
pentachlorophenol, perylene, and sulfur.
11.2.2.3 Set the “dump time” to allow greater than 85% removal of the corn oil and
greater than 85% collection of the phthalate.
11.2.2.4 Set the “collect time” to the peak minimum between perylene and sulfur.
655
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Method 1656
11.2.2.5 Verify the calibration with the calibration solution after every 20 extracts.
Calibration is verified if the recovery of the pentachiorophenol is greater
than 85%. If calibration is not verified, the system shall be recalibrated using
the calibration solution, and the previous 20 samples shall be re-extracted and
cleaned up using the calibrated GPC system.
11.2.3 Extract cleanup: GPC requires that the column not be overloaded. The column specified
in this method is designed to handle a maximum of 0.5 g of high molecular weight
material in a5-mL extract. If the extract is known or expected to contain more than
0.5 g, the extract is split into fractions for GPC and the fractions are combined after
elution from the column. The solids content of the extract may be obtained
gravimetrically by evaporating the solvent from a 50- 1 zL aliquot.
11.2.3.1 FIlter the extract or load through the filter holder to remove particulates.
Load the 5.0-mL extract onto the column.
11.2.3.2 Elute the extract using the calibration data determined in Section 11.2.2.
Collect the eluate in a clean 400- to 500-mL beaker.
11.2.3.3 Rinse the sample loading tube thoroughly with methylene chloride between
extracts to prqare for the next sample.
11.2.3.4 If a particularly dirty extract is encountered, a 5.0-mL methylene chloride
blank shall be run through the system to check for carry-over.
11.2.3.5 Concentrate the extract and exchange into hexane per Sections 10.5 and 10.6.
Adjust the final volume to 5.0 mL.
11.3 Solid-phase extraction (SPE).
11.3.1 Setup.
11.3.1.1 Attach the Vac-elute manifold to a water aspirator or vacuum pump with the
trap and gauge installed between the manifold and vacuum source.
11.3.1.2 Place the SPE cartridges in the manifold, turn on the vacuum source, and
adjust the vacuum to 5 to 10 psia.
11.3.2 Cartridge w shii!g: Pre-elute each cartridge prior to use sequentially with 10-mL
portions each of hexane, methanol, and water using vacuum for 30 seconds after each
eluaz*. Follow this pre-elution with I mL methylene chloride and three 10-mL portions
of thó elution solvent (Section 6.6.2.2) using vacuum for 5 minutes after each eluant.
Tap the cartridge lightly while under vacuum to dry between eluants. The three portions
of elution solvent may be collected and used as a blank if desired. Finally, elute the
cartridge with 10 mL each of methanol and water, using the vacuum for 30 seconds after
11.3.3 Cartridge certification: Each cartridge lot must be certified to ensure recovery of the
onmpounds of interest and removal of 2,4,6-trichiorophenol.
11.3.3.1 To make the test mixture, add the trichiorophenol solution (Section 6.6.2.1)
tu the combined calibration standard (Section 6.11). Elute the mixture using
the pmcedurein Section 11.3.4.
656
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M•thod 1656
11.3.3.2 Concentrate the eluant to 1.0 mL and inject 1.0 L of the concentrated eluant
Into the GC using the procedure In SectIon 13. The recovery of all analytes
(including the unresolved OC peaks) shall be within the ranges for recovery
specified in Table 4, and the peak for trichlorophenol shall not be detectable;
otherwise the SPE cartridge Is not performing properly and the cartridge lot
shall be rejected.
11.3.4 Extract cleanup.
11.3.4.1 After cartridge washing (Section 11.3.2), release the vacuum and place the
rack containing the 50-mL volumetric flasks (Section 5.6.2.4) in the vacuum
m*nifold. Re-establish the vacuum at 5 to 10 psia.
11.3.4.2 Using a pipette or a 1-niL syringe, transfer 1.0 niL of extract to the SPE
cartrld e. Apply vacuum for 5 minutes to dry the cartridge. Tap gently to
aid in drying.
11.3.4.3 Elute each cartridge into its volumetric flask sequentially with three 10-niL
portions of the elution solvent (Section 6.6.2.2), using vacuum for 5 minutes
after each portion. Collect the eluants in the 50-niL volumetric flasks.
11.3.4.4 Release the vacuum and remove the 50-niL volumetric flasks.
11.3.4.5 Concentrate the eluted extracts to 1.0 niL using the nitrogen blow-down
apparatus.
11.4 Florisil column.
11.4.1 Place a weight of Florisil (nominally 20 g) predetermined by calibration (Section 7.5) in
a chromatographic column. Tap the column to settle the Florisil and add 1 to 2 cm of
anhydrous sodium sulfate to the top.
11.4.2 Add 60 mL of hexane to wet and rinse the sodium sulfate and Florisil. Just prior to
exposure of the sodium sulfate layer to the air, stop the elution of the hexane by closing
the stopcock on the chromatographic column. Discard the eluate.
11.4.3 Transfer the concentrated extract (Section 10.6.2) onto the column. Complete the
transfer with two 1-mL hexane rinses.
11.4.4 Place a clean 500-mL K-D flask and concentrator tube under the column. Drain the
column into the flask until the sodium sulfate layer is nearly exposed. Elute Fraction 1
with 200 mL of 6% ethyl ether in hexane (WV) at a rate of approximately 5 mL/min.
Remove the K-D flask. Elute Fraction 2 with 200 mL of 15% ethyl ether in hexane
(\TIV) into a second K-D flask. Elute Fraction 3 with 200 mL of 50% ethyl ether in
hexane (V/V).
11.4.5 Concentrate the fractions as in Section 10.6, except use hexane to prewet the column.
Readjust the final volume to 5 or 10 niL as in Section 10.6, depending on whether the
extract was subjected to GPC cleanup, and analyze by gas chromatography per the
procedure in Section 12.
11.5 Alumina column.
11.5.1 Reduce the volume of the extract to 0.5 mL and bring to 1.0 niL with acetone.
657
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Method 1656
1 1 .5.2 Add 3 g of activity Ill neutral alumina to a 10-mL chromatographic column. Tap the
column to settle the alimuna .
11.5.3 Transfer the extract to the top of the column and collect the eluate in a clean lO-mL
concentrator tube. Rinse the extract container with 1 to 2 mL portions of hexane (to a
total volume of 9 inL) and add to the alumina column. Do not allow the column to go
dry.
11.5.4 Concentrate the extract to 1.0 mL if sulfur is to be removed, or adjust the final volume
to 5 or lOniL as in Section 10.6, depending on whether the extract was subjected to
GPC cleanup, and analyze by gas chromatography per Section 13.
Sulfur removal: Elemental sulfur will usually elute entirely in Fraction I of the Florisil column
cleanup.
11.6.1 Transfer the concentrated extract into a clean concentrator tube or FFFE-sealed vial.
Add 1 to 2 drops of mercury or 100 mg of activated copper powder and seal
(Reference 8). If TM sulfite is used, add 1 mL of the TBA sulfite reagent and 2 mL
°° °°i
116.2 Agitatethecontentsofthevialfor I to2hoursonareciprocal shaker. If the mercury
or copper appears shiny, or if precipitated sodium sulfite crystals from the TBA sulfite
reagent are pres *, and if the color reanaina unchanged, all sulfur has been removed; if
not repeat the addition and shaking.
11.6.2.1
If mercury or copper is used, centrifuge and filter the extract to remove all
residual mercury or copper. Dispose of the mercury waste properly. Bring
the final volume to 1.0 mL and analyze by gas chromatography per the
procedure in Section 13.
11.6.2.2 If TBA sulfite is used, add 5 mL of reagent water and shake for I to
2 minutes. Centrifuge and filter the extract to remove all precipitate.
Transfer the hexane (top) layer to a sample vial and adjust the final volume
to 5 or 10 mL as in Section 10.6, depending on whether the extract was
subjected to GPC cleanup, and analyze by gas chromatography per
Section 12.
12. GAsCHROA&4ToeR Hv
Table 2 i& .imnarl therecommended operating conditions for the gas chromatograph. Included in these
tables are the ret on times and minnium levels that can be achieved under these conditions. Examples
of the separations achieved by the primary and confirmatory columns are shown in Figure 2.
12.1 Calibrate the system as described in Section 7.
12.2 Setthe auto-san ler to inject the same volume that was chosen for calibration (Section 7.3.1.3)
for all standards and extracts of blanks and samples.
12.3 Set the data system or GC control to start the temperature program upon sample injection, and
begin data collection after the solvent peak elutes. Set the data system to stop data collection after
the lant analyte is expected to elute and to return the column to the initial temperature.
11.6
658
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Method 1656
13. SYSTEM AND LABORATORY PERFORMANCE
13.1 At the beginning of each 8-hour shift during which analyses are performed, GC system
performance and calibration are verified for all pollutants and surrogates on both column/detector
systems. For these tests, analysis of the combined QC standard (Section 6.11) shall be used to
verify all performance criteria. Adjustment and/or recalibration (per Section 7) shall be performed
until all performance criteria are met. Only after all performance criteria are met may samples,
blanks, and precision and recovery standards be analyzed.
13.2 Retention times: The absolute retention times of the peak maxima shall be within ±10 seconds
of the retention times in the initial calibration (Section 7.4.1).
13.3 OC resolution: Resolution is acceptable if the valley height between two peaks (as measured from
the baseline) is less than 10% of the taller of the two peaks.
13.3.1 Primary column (DB-608): DDT and endrin aldehyde.
13.3.2 Confirmatory column (DB-1701): Alpha and gamma chlordane.
13.4 Decomposition of DDT and endrin.
13.4.1 Analyze a total of 2 ng DDT and 1 ng endrin on each column using the analytical
conditions specified in Table 2.
13.4.2 Measure the total area of all peaks in the chromatogram.
13.4.3 The area of peaks other than the sum of the areas of the DDT and endrin peaks shall be
less than 20% the sum of the areas of these two peaks. If the area is greater than this
sum, the system is not performing acceptably for DDT and endrin. In this case, the GC
system that failed shall be repaired and the performance tests (Sections 13.1 to 13.4) shall
be repeated until the specification is met.
NOTE: DDT and endrin decomposition are usually caused by accwnulations of
particulates in the injector and in the front end of the colwnn. aeaiung and silanizing the
injection port liner, and breaking off a short section of the front end of the column will
usually eliminate the decomposition problem.
13.5 Calibration verification: Calibration is verified for the combined QC standard only.
13.5.1 Inject the combined QC standard (Section 6.11)
13.5.2 Compute the percent recovery of each compound or coeluting compounds, based on the
calibration data (Section 7.4).
13.5.3 For each compound or coeluted compounds, compare this calibration verification
recovery with the corresponding limits for ongoing accuracy in Table 4. For coeluting
compounds, use the coeluted compound with the least restrictive specification (the widest
range). If the recoveries for all compounds meet the acceptance criteria, system
performance is acceptable and analysis of blanks and samples may begin. If, however,
any recovery falls outside the calibration verification range, system performance is
unacceptable for that compound. In this case, correct the problem and repeat the test,
or recalibrate (Section 7). If verification requirements are met, the calibration is assumed
to be valid for the multicomponent analytes (PCBs and toxaphene).
659
-------
Method 1656
13.6 Ongoing precision and recovery.
13.6.1 Analyze the extract of the precision and recovery standard extracted with each sample
batch (Sections 10.2.3.3 and 10.2.5.7).
13.6.2 Compute the percent recovery of each analyte and for coeluting compounds.
13.6.3 For each compound or coeluted compound, compare the percent recovery with the limits
for ongoing recovery in Table 4. For coeluted compounds, use the coeluted compound
with the least restrictive specification (widest range). If all analytes pass, the extraction,
concentration, and cleanup processes are in control and analysis of blanks and samples
may proceed. If, however, any of the analytes fail, these processes are not in control.
In this event correct the problem, re-extract the sample batch, and repeat the ongoing
precision and recovery test.
13.6.4 Add results which pass the specifications in Section 13.6.3 to initial and previous ongoing
data. Update QC charts to form a graphic representation of continued laboratory
performance. Develop a statement of laboratory data quality for each analyte by
calcualting the average percent recovery (R) and the standard deviation of percent
recovery Sf Express the accuracy as a recovery interval from R - 2s to R + 2sf. For
example, if R=95% and the accuracy is 85 to 105%.
14. QUAUTA 71W DETERMINA liON
14.1 Qualisitative determination is accomplished by comparison of data from analysis of a sample or
blank with data from analysis of the shift standard (Section 13.2), and with data stored in the
retention-time and calibration libraries (Section 7.3.2 and 7.3.3.2). Identification is confirmed
when retention time and amounts agree per the criteria below.
14.2 For each coinjxnmd on each column/detector system, establish a retention-time window ±20
seconds on either side of the retention time in the calibration data (Section 7.3.2). For compounds
that have a retention-time curve (Section 7.3.2.2), establish this window as the minimum
-20 seconds and maximum +20 seconds. For the multi-component analytes, use the retention
times of the five largest peaks in the chromatogram from the calibration data (Section 7.3.2).
14.2.1 Compounds not requiring a retention-time calibration curve: If a peak from the analysis
ofasampleorblankiswithinawindOw(aSdefinediflSeCtiOfl14.2)oflthePfl1 Y
column/detector system, it is considered tentatively identified. A teidalively identified
compound is confirmed when (1) the retention time for the compound on the
confirmatory column/detector system is within the retention-time window on that system,
and (2) the computed amounts (Section 15) on each system (primary and confirmatory)
agree within a factor of three.
14.2.2 Comi$xinds requiring a retention-time calibration curve: If a peak from the analysis of
a siunple or blank is within a window (as defined in Section 14.2) on the primary
column/detector system, it is considered tentatively identified. A tentatively identified
compound is confirmed when (1) the retention times on both systems (primary and
confirmatory) are within ±30 seconds of the retention times for the computed amounts
(Section 15), as dd mined by the retention-time calibration curve (Section 7.3.2.2), and
(2) the computed amounts (Section 15) on each system (primary and confirmatory) agree
within a factor of 3.
660
-------
Method 1656
15.
QUANTITATIVE DETERMINATION
15.1
Using the GC data system, compute the concentration of the analyte detected in the extract
(in
micrograms per milliliter) using the calibration factor or calibration curve (Section 7.3.3.2).
15.2
Liquid samples: Compute the concentration in the sample using the following equation:
Equation 4
(V,)
where
Cs = The concentration in the sample, in pgfL
10 = The extract total, in mL
C = The concentration in the extract, in pg/mL
V 5 = The sample extracted, in L
15.3 Solid samples: Compute the concentration in the solid phase of the sample using the following
equation:
Equation 5
C =10 (Cu )
1000(W 5 ) (solids)
where
C 5 = Concentration in the sample, in i.ig/kg
10 = Extract total, in mL
= Concentration in the extract, in pgJmL
1000 = Conversion factor, g to kg
W , = Sample weight, in g
solids = Percent solids in Section 10.1.3 divided by 100
15.4 If the concentration of any analyte exceeds the calibration range of the system, the extract is
diluted by a factor of 10, and a 1-jiL aliquot of the diluted extract is analyzed.
15.5 Two or more PCBs in a given sample are quantitated and reported as total PCB.
15.6 Report results for all pollutants found in all standards, blanks, and samples to three significant
figures. Results for samples that have been diluted are reported at the least dilute level at which
the concentration is in the calibration range.
16. ANAL YSIS OF COMPLEX SAMPLES
16.1 Some samples may contain high levels (greaer than 1000 ng/L) of the compounds of interest,
interfering compounds, and/or polymeric materials. Some samples may not concentrate to 10 mL
(Section 10.6); others may overload the GC column and/or detector.
661
-------
Method 1656
16.2 The analyst shall attempt to clean up all samples using GPC (Section 11.2), the SPE cartridge
(Section 11.3), by Florisil (Section 11.4) or Alumina (11.5), and sulfur removal (Section 11.6).
If these techniques do not remove the interfering compounds, the extract is diluted by a factor of
10 and reanalyzed (Section 15.4).
16.3 Recovery of surrogate: In most samples, surrogate recoveries will be similar to those from
reagent water or from the high solids reference matrix. If the surrogate recovery is outside the
range specified in Section 8.3.3, the sample shall be reextracted and reanalyzed. If the surrogate
recovery is still outside this range, the extract is diluted by a factor of 10 and reanalyzed
(Section 15.4).
16.4 Recovery of matrix spikes: In most samples, matrix spike recoveries will be similar to those from
reagent water or from the high-solids reference matrix. If the matrix spike recovery is outside
the range specified in Table 4, the sample shall be diluted by a factor of 10, respiked, and
reanalyzed. If the matrix spike recovery is still ‘outside the range, the method may not apply to
the sample being analyzed and the result may not be reported for regulatory compliance purposes.
17. METHoD PERFORMANCE
17.1 Development of this method is detailed in References 9 and 10.
662
-------
Method 1656
References
1. “Carcinogens: Working with Carcinogens.” Department of Health, Education, and Welfare;
Public Health Service; Center for Disease Control; National Institute for Occupational Health and
Safety: Publication 77-206, Aug 1977.
2. “OSHA Safety and Health Standards, General Industry” (29 CFR 1910). Occupational Safety and
Health Administration: Jan 1976.
3. “Safety in Academic Chemistry Laboratories,” American Chemical Society Committee on
Chemical Safety: 1979.
4. Mills, P. A., “Variation of Florisil Activity: Simple Method for Measuring Adsorbent Capacity
and Its Use in Standardizing Florisil Columns,” Journal of the Association of Official Analytical
Chemists, 51, 29: 1968.
5. “Handbook of Quality Control in Wastewater Laboratories,” U.S. Environmental Protection
Agency, Environmental Monitoring and SupportLaboratory, Cincinnati, OH,; EPA-600/4-79-019,
March 1979.
6. “Standard Practice for Sampling Water” (ASTM Annual Book of Standards), American Society
for Testing and Materials, Philadelphia, Pennsylvania: 76, 1980.
7. “Methods 330.4 and 330.5 for Total Residual Chlorine,” U.S. Environmental Protection Agency,
Environmental Monitoring and Support Laboratory, Cincinnati, OH: EPA-600/4-70-020, March
1979.
8. Goerlitz, D.F., and Law, L.M., “Bulletin for Environmental Contamination and Toxicology”: 6,
9, 1971.
9. “Consolidated GC Method for the Determination of ITD/RCRA Pesticides using Selective GC
Detectors,” S-CUBED, A Division of Maxwell Laboratories, Inc., La Jolla, CA: Ref. 32145-01,
Document R70, September 1986.
10. “Method Development and Validation, EPA Method 1618, Cleanup Procedures,” Pesticide Center,
Department of Environmental Health, Colorado State University: November 1988 and January
1989.
663
-------
Method 1656
Table 1. Organo-Halide Pesticides Determined by Large-Bore, Fused-Silica
Capillary Column Gas Chromatography with Halide-Specific Detector
EPA
EGO Compound CAS Registry
Acephate 30560-19-1
Acifluorfen 50594-66-6
Alachior 15972-60-8
089 Aldrin 309-00-2
Atrazine 1912-24-9
Benfluralin (Benefin) 1861-40-1
102 a-BHC 319-84-6
103 -BHC 319-85-7
104 ö-BHC (Lindane) 58-89-9
105 y-BHC 319-86-8
Bromadil 314-40-9
Bromoxynil octanoate 1689-99-2
Butachlor 23184-66-9
434 Captafol 2425-06-1
433 Captan 133-06-2
441 Carbophenothion (Trithion) 786-19-6
a-Chlordane (cis-Chlordane) 5103-71-9
091 y-Chlordane (trans-Chiordane) 5566-34-7
431 Ch lorobenzilate 510-15-6
Chioroneb (‘Ferraneb) 2675-77-6
Chioropropylate (Acaralate) 5836- 10-2
Chiorothalonil 1897-45-6
DBCP (Dibromochloropropane) 96-12-8
DCPA (Dacthal) 1861-32-1
094 4,4’-DDD (FDE) 72-54-8
093 4,4’ -DDE 72-55-9
092 4,4’-DDT 50-29-3
432 Diallate (Avadex) 2303-16-4
664
-------
Method 1656
Table 1. Organo-Hailde Pesticides Determined by Large-Bore, Fused-Silica
Capillary Column Gas Chromatography with Halide-Specific Detector,
cont.
EPA
EGD Compound CAS Registry
478 Dichione 117-80-6
Dicofol 115-32-2
090 Dieldrin 60-57-1
095 Endosulfan I 959-98-8
096 Endosulfan II 33213-65-9
097 Endosulfan sulfate 1031-07-8
098 Endrin 72-20-8
099 Endrin aldehyde 7421-93-4
435 Endrin ketone 53494-70-5
Ethalfluralin (Sonalan) 55283-68-6
Etridiazole 2593-15-9
Fenarimol (Rubigan) 60 168-88-9
100 Heptachior 76-44-8
101 Heptachior epoxide 1024-57-3
437 Isodrin 465-73-6
Isopropalin (Paarlan) 33820-53-0
439 Kepone 143-50-0
430 Methoxychior 72-43-5
Metribuzm 21087-64-9
438 Mirex 2385-85-5
436 Nitrofen (TOK) 1836-75-5
Norfluorazon 27314-13-2
112 PCB-1016 12674-11-2
108 PCB-1221 11104-28-2
109 PCB-1232 11141-16-5
106 PCB-1242 53469-21-9
110 PCB-1248 12672-29-6
665
-------
Method 1656
Table 1. Organo-Halide Pesticides Determined by Large-Bore, Fused-Silica
Capillary Column Gas Chromatography with Halide-Specific Detector,
cont.
EPA
EGD Compowid CAS Registry
107 PCB-1254 11097-69-1
111 PCB-1260 11096-82-5
440 PCNB (pentachloronitrobenzene) 82-68-8
Pendamethalin (Prowl) 40487-42-1
cis-Permethrin 61949-76-6
trans-Permethrin 61949-77-7
Perthane (Ethylan) 72-56-0
Propachior 1918-16-7
Propanil 709-98-8
Propazine 139-40-2
Simw ine 122-34-9
Strobane 8001-50-1
Terbacil 5902-51-2
Terbuthylazine 5915-41-3
113 Toxaphene 8001-35-2
Triadimefon (Bayleton) 43121-43-3
442 Trifluralin 1582-09-8
666
-------
Method 1656
Table 2. Gas Chromatography of Organo-Hatide Pesticides
Retention Time (min)(1) Method Detection
EPA LImIt(2)
EGD Compound DB-608 DB- 1701 (ng/L)
Acephate 5.03 (3) 2000 est (ECD)
442 Trifluralin 5.16 6.79 50 est
Ethalfiuralin 5.28 6.49 5 est (ECD)
Benfluralin 5.53 6.87 20 est (ECD)
432 Diallate-A 7.15. 6.23 45
DiaIlate-B 7.42 6.77 32
102 a-BHC 8.14 7.44 6
440 PCNB 9.03 7.58 6
Simazine 9.06 9.29 400 est (ECD)
Atrazine 9.12 9.12 500 est (ECD)
Terbuthylazine 9.17 9.46 300 est (ECD)
104 ö-BHC (Lindane) 9.52 9.91 11
103 -BHC 9.86 11.90 7
100 Heptachior 10.66 10.55 5
Chiorothalonil 10.66 10.96 15 est (ECD)
478 Dichione 10.80 (3) (4)
Terbacil 11.11 12.63 200 est (ECD)
105 7 -BHC 11.20 12.98 5
Alachior 11.57 11.06 20 est (ECD)
Propanil 11.60 14.10
089 Aldrin 11.84 1146 8
DCPA 12.18 12.09 3 est (ECD)
Metribuzin 12.80 11.68 5 est (ECD)
Triadimefon 12.99 13.57 50 est (ECD)
Isopropalin 13.06 13.37 20 est (ECD)
437 Isodrin 13.47 11.12 13
101 Heptacblor epoxide 13.97 12.56 12
Pendamethalin 14.21 13.46 30
667
-------
Method 1656
Table 2. Gas Chromatography of Organo-Halide Pesticides, cont.
Retention Time (min)(1) Method Detection
EPA Limit(2)
EGD Compound DB-608 DB-1701 (ng/Q
Bromadil 14.39 (3) 70 est (ECD)
6-ch lordane 14.63 14.20 9
Butachlor 15.03 15.69 30 est (ECD)
091 a-chJordane 15.24 14.36 8
095 Endosulfan l 15.25 13.87 11
093 4,4’-DDE 16.34 14.84 10
090 Dieldrin 16.41 15.25 6
433 Captan 16.83 15.43 100 est(ECD)
431 Chlorobenzilate 17.58 17.28 25
098 Endrin 17.80 15.86 4
436 Nitrofen FOK) 17.86 17.47 13
439 Kepone 17.92 24.03 100 est (ECD)
094 4,4’-DDD 18.43 17.77 5
096 Endosulfanll 18.45 18.57 8
Bromoxynil octanoate 18.85 18.57 30 est (ECD)
092 4,4’-DDT 19.48 18.32 12
441 Carbophenothion 19.65 18.21 50
099 Endrinaldehyde 19.72 19.18 11
097 Endosulfan sulfate 20.21 20.37 7
434 Captafol 22.51 21.22 100 est (ECD)
Norfluorazon 20.68 22.01 50 est (ECD)
438 Mirex 22.75 19.79 4
430 M hoxychlor 22.80 20.68 30
435 Endr nketone 23.00 21.79 8
Fenariniol 24.53 23.79 20 est (ECD)
cis-Permethrin 25.00 23.59 200 est (ECD)
trans-Perm hrin 25.62 23.92 200 est (ECD)
106 PCB-1242 150 est
668
-------
Method 1656
Table 2. Gas Chromatography of Organo-Halide Pesticides, cont.
Retention Time (min)(1) Method Detection
EPA Li 2
EGD Compound DB-608 DB-1701 (ng /
109 PCB-1232 150 est
112 PCB-1016 150 est
108 PCB-1221 150 est
110 PCB-1248 150 est
107 PCB-1254 150 est
111 PCB-1260 15.44 14.64 140
15.73 15.36
16.94 16.53
17.28 18.70
19.17 19.92
113 Toxaphene 16.60 16.60 910
17.37 17.52
18.!! 17.92
19.46 18.73
19.69 19.00
Notes:
Columns: 30 m long by 0.53 mm ID; DB-608: 0.83 j ; DB-1701: 1.0 . Conditions suggested
to meet retention times shown: 150°C for 0.5 minutes, 150 to 270° at 5°C/mm, 270°C until
trans-permethrin elutes. Carrier gas flow rates approximately 7 mL/min.
2. 4.0 CFR Part 136, Appendix B (49 FR 43234). MDL’s were obtained with an electrolytic
conductivity detector, except as noted. MDL’S for soils (in ng/kg) are estimated to be 30 to 100
times this level.
3. Does not elute from DB -1701 column at level tested.
4. Not recovered from water at the levels tested.
669
-------
Method 1656
Table 3. Concentrations of Calibration Solutions for Electron Capture Detector
and Suggested Calibration Groups
Concentration (ny/mU
EPA
EGD Compowid Low Medium High
Calibration group 1
Acephate 2000 10000 40000
Alachlor 20 100 400
Atrazlne 1000 5000 20000
1O3 -BHC 10 50 200
Broinoxynhl octanoate 50 250 1000
434 Captalbl 200 1000 4000
432 Diallate 200 1000 4000
097 Endosulfan Sulfate 10 50 200
098 Endrin - 20 100 400
437 Isodrin 10 50 200
Pendimethalin (Prowl) 50 250 1000
trans-Permethrin 200 1000 4000
Calibration group 2
102 a-BHC 5.0 25 100
DCPA 5.0 25 100
093 4,4’-DDE 10 50 200
092 4,4’-DDT 10 50 200
478 Didilone 20 100 400
Ethiffluralin 10 50 200
Fenarimol 20 100 400
430 Metboxychlor 20 100 400
Metrlbuzin 10 50 200
Calibration group 3
1 05 7 -BLIC 5 25 100
091 a-Chlordane 5 25 100
435 Endrinketone 10 50 200
101 Heptachlor epoxide 5 25 100
670
-------
Method 1656
Table 3. Concentrations of Calibration Solutions for Electron Capture Detector
and Suggested Calibration Groups, cont.
Concentration (ng/mL)
EPA
EGD Compound Low Medium High
Isopropalin 20 100 400
436 Nitrofen (FOK) 20 100 400
440 PCNB 5 25 100
cis-Permethrin 200 1000 4000
442 Trifluralin 10 50 200
Calibration group 4
Benfluralin 20 100 400
431 Chlorobenzilate 50 500 5000
090 Dieldrin 5 20 100
095 Endosulfan 1 10 50 200
438 Mirex 20 100 400
Terbacil 200 1000 4000
Terbuthylazine 500 2500 10000
Triadimefon 100 500 2000
Cal ibration group 5
a-Chlordane 10 50 200
433 Captan 100 500 2000
Chlorothalonil 20 100 400
094 4,4’-DDD 20 100 400
Norfluorazon 100 500 2000
Simazine 800 4000 20000
Calibration group 6
089 Aldrin 20 100 400
104 6-BHC (Lindane) 5 25 100
Bromacil 100 500 2000
Butachlor 50 250 1000
096 Endosulfan 11 100 500 2000
671
-------
Method 1656
Table 3. Concentrations of Calibration Solutions for Electron Capture Detector
and Suggested Calibration Groups, cont.
Concentration (ng/mL)
EPA
EOD Cwnpowid Low Medium High
lOOHeptadilor 10 50 200
439 Kepone 100 500 2000
672
-------
Method 1656
Table 4. Acceptance Criteria for Performance Tests for Organo-Halide
Compounds
EGO
No. Compound
Acephate
Alachior
089 AIdrin
Atrazine
Benfluralin
102 a-BHC
103 fl-BHC
105 ô-BHC
104 7 -BHC (Lindane)
Bromacil
Bromoxynil octanoate
Butachior
434 Captafol
433 Captan
441 Carbophenothion
091 Chlordane-a
Ch1ordane-
431 Chlorobenzil ate
Chiorothalonil
DCPA
094 4,4’-DDD
093 4,4’-DDE
092 4,4’-DDT
432 Diallate
478 Dichlone
090 Dieldrin
Spike
Level Precision Verifi-
(ng/L) and Accuracy
S X
100000 94 0-195
1000 20 26 -100
1000 12 82 -108
50000 26 35-129
1000 22 45-125
250 10 57 - 135
500 10 66-130
250 24 60-122
250 10 66-112
5000 84 0 -263
2500 28 31 - 131
2500 32 21 - 137
10000 76 0 -221
5000 32 28-144
1000 10 63 -141
500 10 79-122
250 13 32 -140
500 19 58 -118
1000 20 37-109
250 20 57 - 129
1000 12 69-117
500 13 66-114
500 19 86-112
1000 16 44-120
1000 20 45 -117
500 11 66 -140
cation *
(96)
Acceptance Criteria
Initial Calibration Recovery
Ongoing
Accuracy
R(%)
0 - 209
23 - 101
76 - 114
31 - 132
42 - 128
38 - 154
50 - 146
45 - 136
55 - 123
0 - 275
27 - 135
17 - 141
0 - 232
24 - 148
43 - 161
69 - 133
4 - 169
43 - 133
34 - 112
54 - 132
57 - 129
54 - 126
79 - 119
24 - 139
42 - 120
48 - 158
6 - 194
80 - 120
79 - 113
74 - 126
78 - 122
69 - 108
85 - 102
79 - 103
75 - 119
16 - 184
72 - 128
68 - 132
24 - 176
49 - 114
79 - 102
73 - 102
79 - 113
54 - 129
80 - 120
80 - 120
77 - 109
81 - 121
77 - 118
70 - 124
79 - 110
48 - 115
673
-------
Method 1656
Table 4. Acceptance Criteria for Performance Tests for Organo-Halide
Compounds, cont.
EGD
M. Compound ________ _____
095 Endosulfan I
096 Endosulfan II
097 Endosulfan sulfate
098 End n
099 Endrin aldehyde
435 Endrin ketone
Ethalfiuralin
Fenarimol
100 Heptachlor
101 Heptadilor epoxide
437 Isodrin
‘sop—in
439 Kepone
430 Methoxychior
Metribuzin
438 Mlrex
436 Nitrofen (FOK)
Norfluorazon
112 PCB-1016
108 PCB-1221
109 PCB-1232
106 PCB-1242
110 PCB-1248
107 PCB-1254
111 PCB-1260
Acceptance Criteria
Spèke Initial Calibration Recove,y
Level Precision Verifi- Ongoing
(ng/L) and Accuracy cation’ Accuracy
s X (%) R(%)
500
500
500
1000
1000
500
500
1000
500
250
500
1000
5000
200
500
1000
1000
5000
14 41 -133
19 78-142
17 50-130
13 17-149
13 0-149
25 36-126
24 46-132
26 46 -140
12 78 -104
13 63-117
15 69-113
20 47-129
46 31 -197
19 50-136
24 54-140
23 25-155
22 15 - 139
20 71 -143
78 - 119
76 - 119
70 - 109
5 - 117
86 - 117
68 - 135
76 - 124
74 - 126
80 - 114
79 - 117
71 - 126
80 - 120
47 - 134
47 - 128
76 - 124
78 - 114
59 - 142
80 - 120
18 - 156
62 - 158
31 - 149
0 - 182
0 - 190
14 - 148
42 - 136
42 - 144
71 - 111
49 - 131
45 - 127
54 - 132
25 - 203
28 - 158
50 - 155
0 - 188
0 - 170
68-146
1000 20 82 -112
79-126 75 - 119
674
-------
Method 1656
Table 4. Acceptance Criteria for Performance Tests for Organo-Hailde
Compounds, cont.
Acceptance Criteria
EGO
No. Compound
Spike Initial
Level Precision
(ng/L) and Accuracy
Calibration Recovery
Verifi- Ongoing
cation* Accuracy
(%) R (96)
* Verified at the level of the median standard in Table 3.
S X
440
PCNB
250
11
49
- 129
78
- 101
29
-
149
Pendamethalin
2500
24
32
- 118
76
- 124
28
-
122
cis-Permethrin
10000
30
45
- 153
70
- 130
41
-
157
trans-Permethrin
10000
20
59
- 131
80
- 120
56
-
134
Simazine
40000
20
16
- 100
80
- 120
13
-
101
Terbadil
10000
82
0
- 217
18
- 182
0
-
228
113
Terbuthylazine
Toxaphene
Triadimefon
25000
5000
5000
20
20
54
32
82
32
- 104
- 112
- 104
80
68
80
- 120
- 134
- 120
29
76
0
-
-
-
107
122
107
442
Trifluralin
500
12
32
- 148
47
- 134
3
-
177
675
-------
Method 1656
<30% Solids
Di i To 1% Solids
Concentrate
676
Percent Solids
___ > 30% Solids
ACN & CH 2 CI 2 Sonic
CH 2 Cl 2 UqJUq. Ext. H 2 0 Back-Extract
Concentrate
I I
To Cleanup To Cleanup
Extraction and Concentraction Steps
From Extraction
Gel Permeation
Solid Phase Ext.
Flonsil
Remove Sulfur
GC/HSD
Cleanup and Analysis Steps
Figure 1. Extraction, Cleanup, Derivatization, and Analysis
-------
(B)
0
IIIIIlIIIIItIIIIIIIII 111111 {IIIIlIIIII...uII . .III
0
0
(A)
0
3
I T
I
567 8
Method 1656
• IuIIIIuuuuIIuuuuIIIIIuIflhuIuuIuuuuII(lIuII,IIIuII,uIuulJuuIIuIuIIlIuI
9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25
Retention Time (minutes)
Figure 2. Gas Chromatogram of Selected Organo-Chionne Compounds.
0
0
0
0
0
a
2
0 0
Ca
• IllI ul ulIl iu .uuu.u.ui. uuuuuIIuIuuuuuuuuI IuuuuI uu IuIuIIuuuI 1,111111111
0
0
C
A52-O -87
677
-------
Method 1657
The Determination of
Organo-Phosphorus Pesticides
in Municipal and Industrial
Waste water
-------
Method 1657
The Determination of Organo-Phosphorus Pesticides in Municipal and
Industrial Wastewater
1. SCOPE AND APPLICA TION
1.1 This method is designed to meet the survey requirements of the Environmental Protection Agency
(EPA). It is used to determine the (1) organo-phosphorus pesticides associated with the Cl ean
Water Act, the Resource Conservation and Recovery Act, and the Comprehensive Environmental
Response, Compensation and Liability Act; and (2) other compounds amenable to extraction and
analysis by automated, wide-bore capillary column gas chromatography (CC) with a flame
photometric detector.
1.2 The compounds listed in Table 1 may be determined in waters, soils, sediments, and sludges by
this method. The method is a consolidation of several EPA methods. For waters, the sample
extraction and concentration steps are essentially the same as in these methods. However, the
extraction and concentration steps have been extended to other sample matrices. The method may
be applicable to other phosphorus containing pesticides. The quality assurance/quality control
requirements in this method give the steps necessary to determine this applicability.
1.3 This method is applicable to a large number of compounds. Calibrating the CC systems for all
compounds is time-consuming. If only a single compound or small number of compounds is to
be tested for, it is necessary to calibrate the CC systems and meet the performance specifications
in this method for these compounds only. In addition, the CC conditions can be optimized for
these compounds provided that all performance specifications in this method are met.
1.4 When this method is applied to analysis of unfamiliar samples, compound identity must he
supported by at least one additional qualitative technique. This method describes analytical
conditions for a second gas chromatographic column that can he used to confirm measurements
made with the primary column. Gas chromatography/mass spectrometry (GC/MS) can be used
to confirm compounds in extracts produced by this method when analyte levels are sufficient.
1.5 The detection limits of this method are usually dependent on the level of interferences rather than
instrumental limitations. The limits in Table 2 typify the minimum quantities that can be detected
with no interferences present.
1.6 This method is for use by or under the supervision of analysts experienced in the use of a gas
chromatograph and in the interpretation of gas chromatographic data. Each laboratory that uses
this method must demonstrate the ability to generate acceptable results using the procedure in
Section 8.2.
2. SUMMARY OF METHOD
2.1 Extraction.
2.1.1 The percent solids content of a sample is determined.
2.1.2 Aqueous samples containing less than or equal to 1% solids.
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Method 1657
2.1.2.1 Samples containing water-insoluble compounds: A 1-L sample is extracted
with methylene chloride using continuous extraction techniques.
2.1.2.2 Samples containing highly water-soluble compounds such as methamidophos:
Salt is added to a 1-L sample and the sample is extracted with an azeotropic
mixture of chloroform:acetone using continuous extraction techniques.
2.1.3 Samples containing greater than 1% solids:
2.1.3.1 Non-sludge samples: If the solids content is 1 to 30%, the sample is diluted
to 1% solids with reagent water, homogenized ultrasonically, and extracted
as an aqueous sample. If the solids content is greater than 30%, the sample
is extracted with methylene chloride:acetone using ultrasonic techniques.
21.3.2 Municipal sludge samples and other intractable sample types: If the solids
content is less than 30%, the sample is diluted to 1% solids and extracted as
an aqueous sample. If the solids content is greater than 30%, the sample is
extracted with acetonitrile and then methylene chloride using ultrasonic
techniques. The extract is back-extracted with 2% (W/V) sodium sulfate in
reagent water to remove water-soluble interferences and residual acetonitrile.
2.2 Concentration and cleanup: Each extract is dried over sodium sulfate, concentrated using a
Kuderna-Danish evaporator 1 cleaned up (if necessary) using gel permeation chromatography
(GPC) and/or solid-phase extraction, and concentrated to I mL.
2.3 Gas chrpmatography: A fixed volume of the extract is injected into the gas chromatograph (GC).
The compounds are separated on a wide-bore, fused-silica capillary column and detected using a
flame photometric detector.
2.4 IdentifIcation of a pollutant (qualitative analysis) is performed by comparing the GC retention
times of the compound on two dissimilar columns with the respective retention times of an
authentic standard. Compound identity is confirmed when the retention times agree within their
respective windows.
2.5 Quantitative analysis is performed by using an authentic standard to produce a calibration factor
or calibration curve, and using the calibration data to determine the concentration of a pollutant
in the extract. The concentration in the sample is calculated using the sample weight or volume
and the extract volume.
2.6 Quality is assured through reproducible calibration and testing of the extraction and GC systems.
3. CON TA MINA 710N AND INTERFERENCES
3.1 Solvents, reagents, glassware, and other sample-processing hardware may yield artifacts and/or
elevated baselines causing misinterpretation of chromatograms. All materials used in the analysis
shall be demonstrated to be free from interferences under the conditions of analysis by running
method blanks as described in Section 8.5.
3.2 Glassware and, where possible, reagents are cleaned by rinsing with solvent and baking at 450°C
for 1 hour minimum in a muffle furnace or kiln. Some thermally stable materials, such as PCBs,
may not be eliminated by this treatment and thorough rinsing with acetone and pesticide-quality
hexane may be required.
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Method 1657
3.3 Specific selection of reagents and purification of solvents by distillation in all-glass systems may
be required.
3.4 Interferences coextracted from samples will vary considerably from source to source, depending
on the diversity of the site being sampled. The cleanup procedures given in this method can be
used to overcome many of these interferences, but unique samples may require additional cleanup
to achieve the minimum levels given in Table 2.
4. SAFETY
4.1 The toxicity or carcinogenicity of each compound or reagent used in this method has not been
precisely determined; however, each chemical compound should be treated as a potential health
hazard. Exposure to these compounds should be reduced to the lowest possible level. The
laboratory is responsible for maintaining a current awareness file of OSHA regulations regarding
the safe handling of the chemicals specified in this method. A reference file of material handling
sheets should also be made available to all personnel involved in these analyses. Additional
information on laboratory safety can be found in References I to 3.
4.2 Unknown samples may contain high concentrations of volatile toxic compounds. Sample
containers should be opened in a hood and handled with gloves that will prevent exposure. The
oven used for sample drying to determine percent moisture should be located in a hood so that
vapors from samples do not create a health hazard in the laboratory.
5. APPARA fl/S AND MA TEPJALS
NOTE: Brand names, suppliers, and part numbers are for illustrative purposes only. No
endorsement is implied. Equivalent performance may be achieved using apparatus and
materials other than those spec j/ied here, but demonstration of equivalent perfonnance
meeting the requirements of this method is the responsibility of the laboratory.
5.1 Sampling equipment for discrete or composite sampling.
5.1.1 Sample bottles and caps.
5.1.1.1 Liquid samples (waters, sludges and similar materials that contain less than
5% solids): Sample bottle, amber glass, 1-L or 1-quart, with screw-cap.
5.1.1.2 Solid samples (soils, sediments, sludges, filter cake, compost, and similar
materials that contain greater than 5% solids): Sample bottle, wide-mouth,
amber glass, 500-mL minimum.
5.1.1.3 If amber bottles are not available, samples shall be protected from light.
5.1.1.4 Bottle caps: Threaded to fit sample bottles. Caps shall be lined with PTFE.
5.1.1.5 Cleaning.
5.1.1.5.1 Bottles are detergent-water washed, then rinsed with solvent or
baked at 450°C for 1 hour minimum before use.
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Method 1657
5.1.1.5.2 Liners are detergent water washed, then rinsed with reagent water
and solvent, and baked at approximately 200°C for 1 hour
minimum prior to use.
5.1.2 Compositing equipment: Automatic or manual compositing system incorporating glass
containers cleaned per bottle cleaning procedure above. Sample containers are kept at
0 to 4°C during sampling. Glass or PTFE tubing only shall be used. If the sampler uses
a peristaltic pump, a minimum length of compressible silicone rubber tubing may be used
in the pump only. Before use, the tubing shall be thoroughly rinsed with methanol,
followed by repeated rinsings with reagent water to minimize sample contamination. An
integrating flow meter is used to collect proportional composite samples.
5.2 Equipment for determining percent moisture.
5.2.1 Oven, capable of maintaining a temperature of 110°C (±5°C).
5.2.2 Dessicator.
5.2.3 Crucibles, porcelain.
5.2.4 Weighing pans, aluminum.
5.3 Extraction equipment.
5.3.1 Equipment for ultrasonic extraction.
5.3.1.1 Sonic disruptor: 375 watt with pulsing capability and ½- or %-inch disruptor
horn (Ultrasonics, mc, Model 375C, or equivalent).
5.3.1.2 Sonabox (or equivalent): For use with disruptor.
5.3.2 Equipment for liquid-liquid extraction.
5.3.2.1 Continuous liquid-liquid extractor: PTFE or glass connecting joints and
stopcocks without lubrication, 1.5- to 2-L capacity (Hershberg-Wolf
Extractor, Cal-Glass, Costa Mesa, California, 1000- or 2000-mL continuous
extractor, or equivalent).
5.3.2.2 Round-bottom flask: 500-mL, with heating mantle.
5.3.2.3 Condenser: Graham, to fit extractor.
5.3.2.4 pH meter: With combination glass electrode.
5.3.2.5 pH paper: Wide-range (Hydrion Papers, or equivalent).
5.3.3 Separatory funnels: 250-, 500-, 1000-, and 2000-mL, with PTFE stopcocks.
5.3.4 FIltration apparatus.
5.3.4.1 Glass powder funnels: 125- to 250-mL.
5.3.4.2 Filter paper for above (Whatman 41, or equivalent).
5.3.5 Beakers.
5.3.5.1 1.5- to 2-L, calibrated to 1 L.
5.3.5.2 400-to 500-mL.
5.3.6 Spatulas: stainless steel or PTFE.
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Method 1657
5.3.7 Drying column: 400 mm long by 15 to 20 mm ID Pyrex chromatographic column
equipped with coarse glass fit or glass wool plug.
5.3.7.1 Pyrex glass wool: Extracted with solvent or baked at 450°C for 1 hour
minimum.
5.4 Evaporation/concentration apparatus.
5.4.1 Kuderna-Danish (K-D) apparatus.
5.4.1.1 Evaporation flask: 500-mL (Kontes K-570001-0500, or equivalent), attached
to concentrator tube with springs (Kontes K-662750-0012).
5.4.1.2 Concentrator tube: 10-mL, graduated (Kontes K-570050-1025, or equivalent)
with calibration verified. Ground-glass stopper (size 19/22 joint) is used to
prevent evaporation of extracts.
5.4.1.3 Snyder column: l’hree-bail macro (Kontes K-503000-0232, or equivalent).
5.4.1.4 Snyder column: Two-ball micro (Kontes K-469002-0219, or equivalent).
5.4.1.5 Boiling chips.
5.4.1.5.1 Glass or silicon carbide: Approximately 10/40 mesh, extracted
with methylene chloride and baked at 450°C for 1 hour
minimum.
5.4.1.5.2 PTFE (optional):. Extracted with methylene chloride.
5.4.2 Water bath: Heated, with concentric ring cover, capable of temperature control (±2°C),
installed in a fume hood.
5.4.3 Nitrogen-evaporation device: Equipped with heated bath that can be maintained at 35 to
40°C (N-Evap, Organomation Associates, Inc., or equivalent).
5 ,4.4 Sample vials: Amber glass, 1- to 5-mL with PTFE-lined screw- or crimp-cap, to fit GC
autosampler.
5.5 Balances
5.5.1 Analytical: Capable of weighing 0.1 mg.
5.5.2 Top loading: Capable of weighing 10 mg.
5.6 Apparatus for sample cleanup.
5.6.1 Automated gel permeation chromatograph (Analytical Biochemical Labs, Inc. Columbia,
MO, Model GPC Autoprep 1002, or equivalent).
5.6.1.1 Column: 600 to 700 mm long by 25 mm ID, packed with 70 g of SX-3
Bio-beads (Bio-Rad Laboratories, Richmond, CA, or equivalent).
5.6.1.2 Syringe: 10-mL, with Luer fitting.
5.6.1.3 Syringe-filter holder: Stainless steel. Glass fiber or PTFE filters (Gelman
Acrodisc-CR, 1 to 5 micron, or equivalent).
5.6.1.4 UV detectors: 254-mu, preparative or semi-prep flow cell (Isco, Inc., Type
6; Schmadzu, 5 mm path length; Beckman-Altex 152W, 8 j L micro-prep
flow cell, 2 mm path; Pharmacia UV-1, 3 mm flow cell; LDC Milton-Roy
UV-3, monitor #1203; or equivalent).
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Method 1657
5.6.2 Vacuum system and cartridges ibr solid-phase extraction (SPE).
5.6.2.1 Vacuum system: Capable of achieving 0.1 bar Qiouse vacuum, vacuum
pump, or water aspirator), with vacuum gauge.
5.6.2.2 VacElute Manifold (Analytichem International, or equivalent).
5.6.2.3 Vacuum trap: Made from 500-mL sidearm flask fitted with single-hole
rubber stopper and glass tubing.
5.6.2.4 Rack: For holding 50-mL volumetric flasks in the manifold.
5.6.2.5 Column: Mega Bond Elut, Non-polar, C18 Octadecyl, 10 g/60 mL
(Analyticbem International Cat. No. 607H060, or equivalent).
5.7 Centrifuge apparatus.
5.7.1 Centrifuge: Capable of rotating 500-mL centrifuge bottles or 15-mL centrifuge tubes at
5,000 rpm minimum
5.7.2 Centrifuge bottles: 500-mL, with screw-caps, to fit centrifuge.
5.7.3 Centrifuge tubes: 12- to 15-niL, with screw-caps, to fit centrifuge.
5.7.4 Funnel: Buchner, 15 cm.
5.7.4.1 Flask: Filter, for use with Buchner funnel.
5.7.4.2 Filter paper: 15 cm (Whatman #41, or equivalent).
5.8 Mi cel1aneous glassware.
5.8.1 Pipettes: Glass, volumetric, 1-, 5-, and 10-niL.
5.8.2 Syringes: Glass, with Luerlok tip, 0.1-, 1- and 5-niL. Needles for syringes, 2-inch,
22-gauge.
5.8.3 Volumetric f1as s : 10-, 25-, and 50-niL.
5.8.4 Scintillation vials: Glass, 20- to 50-niL, with PTFE-lined screw-caps.
5.9 Gas diromatograph: Shall have splitless or on-column simultaneous automated injection into
sepa*e capillary column.c with a flame photometric detector at the end of each column,
temperature program with isothermal holds, data system capable of recording simultaneous signals
from the two detectors, and shall meet all of the performance specifications in Section 14.
5.9.1 GC colunmc: Bonded-phase fused silica capillary.
5.9.1.1 Primary: 30 m (± 3 m) by 0.5 mm (±0.05 mm) II) DB-1, or equivalent.
5.9.1.2 Confirmatory: DB-1701, or equivalent, with same dimensions as primary
column.
5.9.2 Data system: Shall collect and record GC data, store GC runs on magnetic disk or tape,
process GC data, compute peak areas, store calibration data including retention times and
calibration factors, identify GC peaks through retention times, compute concentrations,
az generate reports.
5.9.2.1 Data acquisition: OC data shall be collected continuously throughout the
analysis and stored on a mass storage device.
5.9.2.2 Calibration factors and calibration curves: The data system shall be used to
record and m2intain lists of calibration factors, and multi-point calibration
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curves (Section 7). Computations of relative standard deviation (coefficient
of variation) are used for testing calibration linearity. Statistics on initial
(Section 8.2) and ongoing (Section 13.6) performance shall be computed and
maintained.
5.9.2.3 Data processing: The data system shall be used to search, locate, identify,
and quantify the compounds of interest in each GC analysis. Software
routines shall be employed to compute and record retention times and peak
areas. Displays of chromatograms and library comparisons are required to
verify results.
5.9.2.4 Flame photometric detector: Capable of detecting 11 pg of malathion under
the analysis conditions given in Table 2.
6. REAGENTS AND STANDARDS
6.1 Sample preservation: Sodium thiosulfate (ACS), granular.
6.2 pH adjustment.
6.2.1 Sodium hydroxide: Reagent grade.
6.2.1.1 Concentrated solution (iON): Dissolve 40 g NaOH in 100 mL reagent water.
6.2.1.2 Dilute solution (0.1M): Dissolve 4 g NaOH in 1 L of reagent water.
6.2.2 Sulfuric acid (1 + 1): Reagent grade, 6N in reagent water. Slowly add 50 mL H 2 S0 4
(specific gravity 1.84) to 50 mL reagent water.
6.2.3 Potassium hydroxide: 37% (W/V); dissolve 37 g KOH in 100 mL reagent water.
6.3 Solution drying and back-extraction.
6.3.1 Sodium sulfate: Reagent grade, granular anhydrous (Baker 3375, or equivalent), rinsed
with methylene chloride (20 mL/g), baked at 450°C for 1 hour minimum, cooled in a
dessicator, and stored in a pre-cleaned glass bottle with screw-cap which prevents
moisture from entering.
6.3.2 Sodium sulfate solution: 2% (WIV) in reagent water, pH adjusted to 8.5 to 9.0 with
KOH or H 2 S0 4 .
6.4 Solvents: Methylene chloride, hexane, acetone, acetonitrile, isooctane, and methanol; pesticide-
quality; lot-certified to be free of interferences.
6.5 GPC calibration solution: Solution containing 300 mglmL corn oil, 15 mg/mL bis(2-ethylhexyl)
phthalate, 1.4 mglmL pentachlorophenol, 0.1 mg/mL perylene, and 0.5 mg/mL sulfur.
6.6 Sample cleanup.
6.6.1 Solid-phase extraction.
6.6.1.1 SPE cartridge calibration solution: 2,4,6-trichiorophenol, 0.1 g/mL in
acetone.
6.6.1.2 SPE elution solvent: methylene chloride:acetonitrile:hexane (50:3:47).
6.7 Reagent water: Water in which the compounds of interest and interfering compounds are not
detected by this method.
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Method 1657
6.8 High-solids reference matrix: Playground sand or similar material in which the compounds of
interest and interfering compounds are not detected by this method. May be prepared by
extraction with methylene chloride and/or baking at 450°C for 4 hours minimum.
6.9 Standard solutions: Purchased as solutions or mixtures with certification to their purity,
concentration, and authenticity, or prepared from materials of known purity and composition. If
compound purity is 96% or greater, the weight may be used without correction to compute the
concentration of the standard. When not being used, standards are stored in the dark at -20 to
-10°C in screw-capped vials with PTFE-lined lids. A mark is placed on the vial at the level of
the solution so that solvent evaporation loss can be detected. The vials are brought to room
temperature prior to use. Any precipitate is redissolved and solvent is added if solvent loss has
oc rred.
6.10 Preparation of stock solutions: Prepare in isooctane per the steps below. Observe the safety
precautions in Section 4.
6.10.1 Dissolve an appropriate amount of assayed reference material in solvent. For example,
weigh 10 mg of malathion in a 10-mL ground-glass stoppered volumetric flask and fill
to the mark with isooctane. After the malathion is completely dissolved, transfer the
solution to a 15-mL vial with FFFE-lined cap.
6.10.2 Stock solutions should be checked for signs of degradation prior to the preparation of
calibration or performance test standards.
6.10.3 Stock standard solutions shall be replaced after 6 months, or sooner if comparison with
quality control check standards indicates a change in concentration.
6.11 Secondary mixtures: Using stock solutions (Section 6.10), prepare mixtures at the levels shown
in Table 3 for calibration and calibration verification (Sections 7.3 and 13.5), for initial and
ongoing precision and recovery (Sections 8.2 and 13.6), and for spiking into the sample matrix
(Section 8.4).
6.12 Surrogate spiking solution: Prepare tributyl phosphate and triphenyl phosphate each at a
concentration of 2 ng/mL in acetone.
6.13 Stability of solutions: All standard solutions (Sections 6.9 to 6.12) shall be analyzed within 48
l un of preparation and on a monthly basis thereafter for signs of degradation. Standards will
rem2in acceptable if the peak area remains within ± 15% of the area obtained in the initial analysis
of the standird .
7. SETUP AND CALIBRATiON
7.1 Configure the GC system as given in Section 5.9 and establish the operating conditions in Table 2.
7.2 AttaInment of method detection limit (MDL): Determine that each column/detector system meets
the MDL’s In Table 2.
7.3 Calibration.
7.3.1 Inj tion of calibration solutions.
7.3.1.1 Compounds with calibration data in Table 3: The compounds in each
calibration group in Table 3 were chosen so that each compound would be
separated from the others by approximately 1 minute on the primary column.
688
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Method 1657
The concentrations were chosen to bracket the working range of the FPD.
However, because the response of some models of FPD are greater than
others, it may be necessary to inject a larger volume of calibration solution
for these detectors.
7.3.1.2 Compounds without calibration data in Table 3: Prepare calibration standards
at a minimum of three concentration levels. One of these concentrations
should be near, but above, the MDL (Fable 2) and the other concentrations
should define the working range of the detectors.
7.3.1.3 Set the automatic injector to inject a constant volume in the range of 0.5 to
5.0 ,zL of each calibration solution into the GC column/detector pairs,
beginning with the lowest level mixture and proceeding to the highest. For
each compound, compute and store, as a function of the concentration
injected, the retention time and peak area on each column/detector system
(primary and confirmatory).
7.3.2 Retention time: The polar nature of some analytes causes the retention time to decrease
as the quantity injected increases. To compensate this effect, the retention time for
compound identification is correlated with the analyte level.
7.3.2.1 If the difference between the maximum and minimum retention times for any
compound is less than 5 seconds over the calibration range, the retention time
for that compound can be considered constant and an average retention time
may be used for compound identification.
7.3.2.2 Retention-time calibration curve (retention time vs. amount): If the retention
time for a compound in the lowest level standard is more than 5 seconds
greater than the retention time for the compound in the highest level standard,
a retention-time calibration curve shall be used for identification of that
compound.
7.3.3 Calibration factor (ratio of area to amount injected).
7.3.3.1 Compute the coefficient of variation (relative standard deviation) of the
calibration factor over the calibration range for each compound on each
column/detector system.
7.3.3.2 Linearity: If the calibration factor for any compound is constant (C < 20%)
over the calibration range, an average calibration factor may be used for that
compound; otherwise, the complete calibration curve (area vs. amount) for
that compound shall be used.
7.4 Combined QC standards: To preclude periodic analysis of all of the individual calibration groups
of compounds (Fable 3), the GC systems are calibrated with combined solutions as a final step.
Not all of the compounds in these standards will be separated by the GC columns used in this
method. Retention times and calibration factors are verified for the compounds that are resolved,
and calibration factors are obtained for the unresolved peaks. These combined QC standards are
prepared at the level the mid-range calibration standard (Table 3).
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Method 1657
7.4.1 Analyze the combined QC standard on each column/detector pair.
7.4.1.1 For those compounds that exhibit a single, resolved OC peak, the retention
time shall be within ±5 seconds of the retention time of the peak in the
medium level calibration standard (Section 7.3.1), and the calibration factor
using the primary column shall be within ±20% of the calibration factor in
the medium level standard (Fable 3).
7.4.1.2 For the peaks containing two or more compounds, compute and store the
retention times at the peak maxima on both columns (primary and
confirmatory), and also compute and store the calibration factors on both
columns. These results will be used for calibration verification (Section 13.2
and 13.5) and for precision and recovery studies (Sections 8.2 and 13.6).
8. QUALITY CONTROL
8.1 Each laboratory that uses this method is required to operate a formal quality control program
(Reference 5). The minimum requirements of this program consist of an initial demonstration of
laboratory capability, an ongoing analysis of standards and blanks as tests of continued
performance, and analysis of spiked samples to assess accuracy. Laboratory performance is
compared to established performance criteria to determine if the results of analyses meet the
performance characteristics of the method. If the method is to be applied routinely to samples
cont$ning high solids with very little moisture (e.g., soils, compost), the high-solids reference
matrix (Section 6.8) is substituted for the reagent water (Section 6.8) in all performance tests, and
the high-solids method (Section 10) is used for these tests.
8.1.1 The analyst shall make an initial demonstration of the ability to generate acceptable
accuracy and precision with this method. This ability is established as described in
Section 8.2.
8.1.2 The analyst is permitted to modify this method to improve separations or lower the costs
of measurements, provided all performance requirements are met. Each time a
modification is made to the method or a cleanup procedure is added, the analyst is
required to repeat the procedure in Section 8.2 to demonstrate method performance. If
detection limits will be affected by the modification, the analyst is required to repeat the
demonstration of detection limits (Section 7.2).
8.1.3 The laboratory shall spike all samples with at least one surrogate compound to monitor
method performance. This test is described in Section 8.3. When results of these spikes
indicate atypical method performance for samples, the samples are diluted to bring
method performance within acceptable limits (Section 16).
8.1.4 The laboratory shall, on an ongoing basis, demonstrate through calibration verification
and the analysis of the combined QC standard (Section 7.4) that the analysis system is
in control. These procedures are described in Sections 13.1, 13.5, and 13.6.
8.1.5 The laboratory shall maintain records to define the quality of data that is generated.
Development of accuracy statements is described in Section 8.4.
8.1.6 Analyses of blanks are required to demonstrate freedom from contamination. The
procedures and criteria for analysis of a blank are described in Section 8.5.
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Method 1657
8.1.7 Other analytes may be determined by this method. The procedure for establishing a
preliminary quality control limit for a new analyte is given in Section 8.6.
8.2 Initial precision and recovery: To establish the ability to generate acceptable precision and
accuracy, the analyst shall perform the following operations.
8.2.1 For analysis of samples containing low solids (aqueous samples), extract, concentrate,
and analyze one set of four 1-L aliquots of reagent water spiked with the combined QC
standard (Section 7.4) according to the procedure in Section 10. Alternatively, sets of
four replicates of the individual calibration groups (Section 7.3) may be used. For
samples containing high solids, sets of four 30 g aliquots of the high-solids reference
matrix are used.
8.2.2 Using results of the set of four analyses, compute the average percent recovery (X) and
the coefficient of variation (Cv) of percent recovery (s) for each compound.
8.2.3 For each compound, compare s and X with the corresponding limits for initial precision
and accuracy in Table 4. For coeluting compounds, use the coeluted compound with the
least restrictive specification (largest C and widest range). Ifs and X for all compounds
meet the acceptance criteria, system performance is acceptable and analysis of blanks and
samples may begin. If, however, any individual s exceeds the precision limit or any
individual X falls outside the range for recovery, system performance is unacceptable for
that compound. In this case, correct the problem and repeat the test.
8.3 The laboratory shall spike all samples with at least one surrogate compound to assess method
performance on the sample matrix.
8.3.1 Analyze each sample according to the method beginning in Section 10.
8.3.2 Compute the percent recovery (P) of the surrogate compounds.
8.3.3 The recovery of the surrogate compounds shall be within the limits of 40 to 120%. If
the recovery of any surrogate falls outside of these limits, method performance is
unacceptable for that sample, and the sample is complex. Water samples are diluted, and
smaller amounts of soils, sludges, and sediments are reanalyzed per Section 16.
8.4 Method accuracy: The laboratory shall spike (matrix spike) at least 10% of the samples from a
given site type (e.g., influent to treatment, treated effluent, produced water, river sediment). If
only one sample from a given site type is analyzed, a separate aliquot of that sample shall be
spiked.
8.4.1 The concentration of the matrix spike shall be determined as follows.
8.4.1.1 If, as in compliance monitoring, the concentration of a specific analyte in the
sample is being checked against a regulatory concentration limit, the matrix
spike shall be at that limit or at 1 to 5 times higher than the background
concentration determined in Section 8.4.2, whichever concentration is larger.
8.4.1.2 If the concentration of an analyte. in the sample is not being checked against
a limit specific to that analyte, the matrix spike shall be at the concentration
of the combined QC standard (Section 7.4) or at 1 to 5 times higher than the
background concentration, whichever concentration is larger.
8.4.1.3 If it is impractical to determine the background concentration before spiking
(e.g., maximum holding times will be exceeded), the matrix spike
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Method 1657
concentration shall be the regulatory concentration limit, if any; otherwise,
the larger of either 5 times the expected background concentration or at the
concentration of the combined QC standard (Section 7.4).
8.4.2 Analyze one sample aliquot to determine the background concentration (B) of each
analyte. If necessary, prepare a standard solution appropriate to produce a level in the
sample one to five times the background concentration. Spike a second sample aliquot
with the standard solution and analyze it to determine the concentration after spiking (A)
of each analyte. Calculate the percent recovery (P) of each analyte:
Equation 1
= 100 ( A-B )
T
where
T = True value of the spike
8.43 Compare the percent recovery for each analyte with the corresponding QC acceptance
criteria in Table 4. If any analyte fails the acceptance criteria for recovery, the sample
is complex and must be diluted and reanalyzed per Section 16.
8.44 As part of the QC program for the laboratory, method accuracy for samples shall be
assessed and records shall be maintained. After the analysis of five spiked samples of
a given matrix type (water, soil, sludge, sediment) in which the analytes pass the tests
in Section 8.4, compute the average percent recovery (P) and the standard deviation of
the percent recovery (s.) for each compound (or coeluting compound group). Express
the accuracy assessment as a percent recovery interval from P - 2s, to P + 2 s for each
matrix. For example, if P = 90% and s = 10% for five analyses of compost, the
accuracy interval is expressed as 70 to 110%. Update the accuracy assessment for each
compound in each matrix on a regular basis (e.g., after each five to ten new accuracy
measurements).
8.5 Blanks: Reagent water and high-solids reference matrix blanks are analyzed to demonstrate
freedom from contamination.
8.5.1 Extract and concentrate a 1 L reagent water blank or a 30 g high-solids reference matrix
blank with each sample batch (samples started through the extraction process on the same
8 hour shift, to a maximum of 20 samples). Analyze the blank immediately after analysis
of the combined QC standard (Section 13.6) to demonstrate freedom from contamination.
8.5.2 If any of the compounds of interest (Table 1) or any potentially interfering compound is
found in an aqueous blank at greater than 0.05 pg/L, or in a high-solids reference matrix
blank at greater than 1 pg/kg (assuming the same calibration factor as malathion for
compounds not listed in Table 1), analysis of samples is halted until the source of
contamination is eliminated and a blank shows no evidence of contamination at this level.
8.6 Other analytes may be determined by this method. To establish a quality control limit for an
analyte, determine the precision and accuracy by analyzing four replicates of the analyte along
with the combined QC standard per the procedure in Section 8.2. If the analyte coelutes with an
692
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Method 1657
analyte In the QC standard, prepare a new QC standard without the coeluting component(s).
Compute the average percent recovery (A) and the standard deviation of percent recovery (sj for
the analyte, and measure the recovery and standard deviation of recovery for the other analytes.
The data for the new analyte is assumed to be valid if the precision and recovery specifications
for the other analytes are met; otherwise, the analytical problem is corrected and the test is
repeated. Establish a preliminary quality control limit of A ± 2s for the new analyte and add
the limit to Table 4.
8.7 The specifications contained in this method can be met if the apparatus used is calibrated properly,
then maintained in a calibrated state. The standards used for calibration (Section 7), calibration
verification (Section 13.5), and for initial (Section 8.2) and ongoing (Section 13.6) precision and
recovery should be identical, so that the most precise results will be obtained. The GC
instruments will provide the most reproducible results if dedicated to the settings and conditions
required for the analyses of the analytes given in this method.
8.8 Depending on specific program requirements, field replicates and field spikes of the analytes of
interest into samples may be required to assess the precision and accuracy of the sampling and
sample transporting techniques.
9. SAMPLE COLLECTION, PRESERVATiON, AND HANDUNG
9.1 Collect samples in glass containers following conventional sampling practices (Reference 6),
except that the bottle shall not be prerinsed with sample before collection. Aqueous samples
which flow freely are collected in refrigerated bottles using automatic sampling equipment. Solid
samples are collected as grab samples using wide-mouth jars.
9.2 Maintain samples at 0 to 4°C from the time of collection until extraction. If the samples will not
be extracted within 72 hours of collection, adjust the sample to a pH of 5.0 to 9.0 using sodium
hydroxide or sulfuric acid solution. Record the volume of acid or base used. If residual chlorine
is present in aqueous samples, add 80 mg sodium thiosulfate per liter of water. EPA Methods
330.4 and 330.5 may be used to measure residual chlorine (Reference 7).
9.3 Begin sample extraction within 7 days of collection, and analyze all extracts within 40 days of
extraction.
10. SAMPLE EXTRACTION AND CONCENTRATION
Samples containing 1% solids or less are extracted directly using continuous liquid-liquid extraction
techniques (Section 10.2.1). Samples containing I to 30% solids are diluted to the 1 % level with reagent
water (Section 10.2.2) and extracted using continuous liquid-liquid extraction techniques. Samples
containing greater than 30% solids are extracted using ultrasonic techniques (Section 10.2.5). For highly
water soluble compounds such as methamidophos, samples are salted and extracted using a
chloroform:acetone azeotrope (Section 10.2.6). Figure 1 outlines the extraction and concentration steps.
10.1 Determination of percent solids.
10.1.1 Weigh 5 to 10 g of sample into a tared beaker. Record the weight to three significant
figures.
10.1.2 Dry overnight (12 hours minimum) at 110°C (± 5°C), and cool in a dessicator.
10.1.3 Determine percent solids as follows:
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Method 1657
Equation 2
% weight of diy sample 100
weight of wet sample
10.2 Preparation of samples for extraction.
10.2.1 Aqueous samples containing 1% solids or less: Extract the sample directly using
continuous liquid-liquid extraction techniques.
10.2.1.1 Measure 1 L (±0.01 L) of sample into a clean 1.5- to 2-L beaker.
10.2.1.2 Spike 0.5 mL of the surrogate spiking solution (Section 6.12) into the sample
aliquot. Proceed to preparation of the QC aliquots for low-solids samples
(Section 10.2.3).
10.22 Samples containing 1 to 30% solids.
10.2.2.1 Mix sample thoroughly.
10.2.2.2 Using the percent solids found in Section 10.1.3, determine the weight of
sample required to produce 1 L of solution containing 1% solids as follows:
Equation3
welht= I000g
10.2.2.3 Place the weight determined in Section 10.2.2 .2 in a clean 1.5-L to 2.0-L
beaker. Discard all sticks, rocks, leaves, and other foreign material prior to
weighing.
10.2.2.4 Bring the volume of the sample aliquot(s) to 100 to 200 mL with reagent
water.
10.2.2.5 Spike 0.5 mL of the appropriate surrogate spiking solution (Section 6.12) into
each sample aliquot.
10.2.2.6 Using a clean metal spatula, break any solid portions of the sample into small
pieces.
10.2.2.7 Place the %-inch horn on the ultrasonic probe approximately ½ inch below
thesurfaceofeachsamplealiquotandpulseat5O% for3 minutes at full
power. If necessary, remove the probe from the solution and break any large
pieces using the metal spatula or a stirring rod and repeat the sonication.
Clean the probe with methylene chloride:acetone (1:1) between samples to
preclude cross-contamination.
102.2.8 Bring the sample volume to 1.0 L (±0.1 L) with reagent water.
10.2.3 Preparation of QC aliquots for samples containing low solids (less than 30%).
694
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Method 1657
10.2.2.5 Spike 0.5 mL of the appropriate surrogate spiking solution (Section 6.12) into
each sample aliquot.
10.2.2.6 Using a clean metal spatula, break any solid portions of the sample into small
pieces.
10.2.2.7 Place the %-inch horn on the ultrasonic probe approximately 1/i inch below
the surface of each sample aliquot and pulse at 50% for 3 minutes at full
power. If necessary, remove the probe from the solution and break any large
pieces using the metal spatula or a stirring rod and repeat the sonication.
Clean the probe with methylene chloride:acetone (1:1) between samples to
preclude cross-contamination.
10.2.2.8 Bring the sample volume to 1.0 L (±0.1 L) with reagent water.
10.2.3 Preparation of QC aliquots for samples containing low solids (less than 30%).
10.2.3.1 For each sample or sample batch (to a maximum of 20) to be extracted at the
same time, place two 1.0 L (±0.01 L) aliquots of reagent water in clean 1.5-
to 2.0-L beakers.
10.2.3.2 Blank: Spike 0.5 mL of the pesticide surrogate spiking solution
(Section 6.12) into one reagent water aliquot.
10.2.3.3 Spike the combined QC standard (Section 7.4) into a reagent water aliquot.
10.2.3.4 If a matrix spike is required,prepare an aliquot at the concentrations specified
in Section 8.4.
10.2.4 Stir and equilibrate all sample and QC solutions for 1 to 2 hours. Extract the samples
and QC aliquots per Section 10.3.
10.2.5 Samples containing 30% solids or greater.
10.2.5.1
Mix the sample thoroughly.
10.2.5.2 Weigh 30 g (±0.3 g) into a clean 400- to 500-mL beaker. Discard all
sticks, rocks, leaves, and other foreign material prior to weighing.
10.25.3 Spike 0.5 mL of the surrogate spiking solution (Section 6.12) into the sample
aliquot.
10.2.5.4 QC aliquots: For each sample or sample batch (to a maximum of 20) to be
extracted at the same time, place two 30 g (±0.3 g) aliquots of the high-
solids reference matrix in clean 400- to 500-mL beakers.
10.2.5.5 Blank: Spike 0.5 mL of the surrogate spiking solution (Section 6.12) into an
aliquot of the high-solids reference matrix.
10.2.5.6 Spike the combined QC standard (Section 7.4) into a high-solids reference
matrix aliquot. Extract the high-solids samples per Section 10.4.
10.2.6 Samples containing methamidophos and other highly water-soluble compounds: Prepare
samples containing less than 30% solids per Sections 10.2.6.1 to 10.2.6.5; prepare
samples containing greater than 30% solids per Section 10.2.5.
10.2.6.1 Interferences: If interferences are expected, aqueous samples can be
pre-extracted with methylene chloride to remove these interferences. This
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Method 1657
extract can be used for determination of insoluble or slightly soluble
compounds and the surrogates. Methamidophos is only sightly soluble in
methylene chloride and will not be in this extract unless carried by polar
species in the sample matrix. If compounds other than methamidophos are
not to be determined, the inethylene chloride extract can be discarded.
10.2.6.2 Determine the percent solids and prepare a 1-L sample aliquot and the QC
aliquots per Sections 10.2.2 and 10.2.4 or 10.2.3 and 10.2.4, except do not
spike the surrogate into the sample aliquot if the methylene chloride extract
will be discarded (Section 10.2.6.1).
10.2.6.3 Extract the aliquots per Section 10.3 using methylene chloride to remove
interferences.
10.2.6.4 After extraction, remove the water and methylene chloride from the extractor.
Decant the aqueous portion into a beaker and combine the remaining
methylene chloride with the extract in the distilling flask. If the methylene
chloride extract is to be used for determination of other analytes and the
surrogate, proceed to Section 10.5 with that extract.
10.2.6.5 Saturate the water sample with sodium chloride. Approximately 350 g will
be required.
10.2.6.6 If the methylene chloride extract was discarded, spike the surrogates into the
sample aliquot.
10.2.6.7 Extract the sample per Section 10.3 except use a chloroform:acetone
azeotrope (2:1 V/V or 4:1 W/W) for the extraction.
NOTE: Note: As a result of the increased density of the water caused by saturation with
salt, the sample may sink to w*ere the water enters the siphon tube of the continuous
extractor. To prevent this from occurring, use a smaller wilume of water (e.g., 8 (X) niL)
in the extractor. Correct jbr this adjustment in the calculation of the concentration of the
pollutant in the extract (Section 15).
10.3 Continuous extraction of low-solids (aqueous) samples: Place 100 to 150 mL methylene chloride
in each continuous extractor and 200 to 300 mL in each distilling flask.
10.3.1 Pour the sample(s), blank, and standard aliquots into the extractors. Rinse the glass
containers with 50 to 100 mL methylene chloride and add to the respective extractors.
Include all solids in the extraction process.
10.3.2 Extraction: Adjust the pH of the waters in the extractors to 5 to 9 with NaOH or H 2 S0 4
while monitoring with a pH meter.
NOTE: Cautlon. Some samples require acid(fication in a hood because of the potential
JEw generating hydrogen sulfide.
6.96
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Method 1657
10.3.3 Begin the extraction by heating the flask until the methylene chloride is boiling. When
properly adjusted. one to two drops of methylene chloride per second will fall from the
condenser tip into the water. Test and adjust the pH of the waters during the first 1 to
2 hours of extraction. Extract fur 18 to 24 hours.
10.3.4 Remove the distilling flask, estimate and record the volume of extract (to the nearest
100 mL), and pour the contents through a prerinsed drying column containing 7 to 10 cm
of anhydrous sodium sulfate. Rinse the distilling flask with 30 to 50 mL of methylene
chloride and pour through the drying column. For extracts to be cleaned up using (iPC,
collect the solution in a 500-mL K-D evaporator flask equipped with a I0-mL
concentrator tube. Seal, label, and concentrate per Sections 10.5 to 10.6.
10.4 Ultrasonic extraction of high-solids samples: Procedures are provided for extraction of
non-municipal sludge (Section 10.4.1) and municipal sludge samples (Section 10.4.2).
10.4.1 Ultrasonic extraction of non-municipal sludge high-solids aliquots.
10.4.1.1
Add 60 to 70 g of powdered sodium sulfate to the sample and QC aliquots.
Mix each aliquot thoroughly. Some wet sludge samples may require more
than 70 g for complete removal of water. All water must be removed prior
to addition of organic solvent so that the extraction process is efficient.
10.4.1.2 Add 100 mL (± 10 mL) of acetone:methylene chloride (1:1) to each of the
aliquots and mix thoroughly.
10.4.1.3 Place the Y -inch horn on the ultrasonic probe approximately ½ inch below
the surface of the solvent hut above the solids layer and pulse at 50% for 3
minutes at full power. If necessary, remove the probe from the solution and
break any large pieces using a metal spatula or a stirring rod and repeat the
sonication.
10.4.1.4 Decant the pesticide extracts through a prerinsed drying column containing
7 to 10 cm anhydrous sodium sulfate into 500- to 1000-mL graduated
cylinders.
10.4.1.5 Repeat the extraction steps (Sections 10.4.1.2 to 10.4.1.4) twice more for
each sample and QC aliquot. On the final extraction, swirl the sample or QC
aliquot, pour into its respective drying column, and rinse with
acetone:methylene chloride. Record the total extract volume. If necessary,
transfer the extract to a centrifuge tube and centrifuge for 10 minutes to settle
fine particles.
10.4.2 Ultrasonic extraction of high solids municipal sludge aliquots.
10.4.2.1 Add 100 mL (±10 mL) of acetonitrile to each of the aliquots and mix
thoroughly.
10.4.2.2 Place the % -inch horn on the ultrasonic probe approximately ½ inch below
the surface of the solvent but above the solids layer and pulse at 50% for
3 minutes at full power. If necessary, remove the probe from the solution
and break any large pieces using a metal spatula or a stirring rod and repeat
the sonication.
697
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Method 1657
10.4.2.3 Decant the extract through filter paper into a 1000- to 2000-mL separatory
funnel.
10.4.2.4 Repeat the extraction and filtration steps (Sections 10.4.2.1 to 10.4.2.3) using
a second 100 mL (±10 mL) of acetonitrile.
10.4.2.5 Repeat the extraction step (Sections 10.4.2.1 and 10.4.2.2) using 100 mL
(± 10 mL) of methylene chloride. On this final extraction, swirl the sample
or QC aliquot, pour into its respective filter paper, and rinse with methylene
chloride. Record the total extract volume.
10.4.2.6 For each extract, prepare 1.5 to 2 L of reagent water containing 2% sodium
sulfate. Adjust the pH of the water to 6.0 to 9.0 with NaOH or H 2 S0 4 .
10.4.2.7 Back-extract each extract three times sequentially with 500 mL of the aqueous
sodium sulfate solution, returning the bottom (organic) layer to the separatory
funnel the first two times while discarding the top (aqueous) layer. On the
final back-extraction, filter each pesticide extract through a prerinsed drying
column containing 7 to 10 cm anhydrous sodium sulfate into a 500- to
1000-mL graduated cylinder. Record the final extract volume.
10.4.3 For extracts to be cleaned up using GPC, filter these extracts through Whatman #41
paper into a 500-mL KD evaporator flask equipped with a 10-mL concentrator tube.
Rinse the graduated cylinder or centrifuge tube with 30 to 50 mL of methylene chloride
and pour through filter to complete the transfer. Seal and label the K-D flask.
Concentrate these fractions per Sections 10.5 through 10.8.
10.5 Macro concentration.
10.5.1 Concentrate the extracts in separate 500-mL K-D flasks equipped with 10-mL
concentrator tubes. Add one to two clean boiling chips to the flask and attach a
three-bali macro Snyder column. Prewet the column by adding approximately one mL
of methylene chloride through the top. Place the K-D apparatus in a hot water bath so
th* the entire lower rounded surface of the flask is bathed with steam. Adjust the
vertical position of the apparatus and the water temperature as required to complete the
concentration in 15 to 20 minutes. At the proper rate of distillation, the balls of the
column will actively chatter but the chambers will not flood.
10.5.2 When the liquid has reached an apparent volume of 1 mL, remove the K-D apparatus
from the bath and allow the solvent to drain and cool for at least 10 minutes.
10.5.3 If the extract is to be cleaned up using GPC, remove the Snyder column and rinse the
flask and its lower joint into the concentrator tube with 1 to 2 mL of methylene chloride.
A 5-mL syringe is recommended for this operation. Adjust the final volume to 10 mL
and proceed to GPC cleanup in Section 11.
10.6 Hexane exchange: Extracts that have been cleaned up are exchanged into hexane.
10.6.1 Remove the Snyder column, add approximately 50 mL of hexane and a clean boiling
diij , and reaft di the Snyder column. Concentrate the extract as in Section 10.5 except
use bexane to prewet the column. The elapsed time of the concentration should be 5 to
10 minutes.
698
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Method 1657
10.6.2 Remove the Snyder column and rinse the flask and its lower joint into the concentrator
tube with 1 to 2 mL of hexane. Adjust the final volume of extracts that have not been
cleaned up by GPC to 10 mL and those that have been cleaned up by GPC to 5 mL (the
difference accounts for the 50% loss in the GPC cleanup).
11. CLEANUP AND SEPARA TION
11.1 Cleanup procedures may not be necessary for relatively clean samples (treated effluents, ground
water, drinking water). If particular circumstances require the use of a cleanup procedure, the
analyst may use any or all of the procedures below or any other appropriate procedure. However,
the analyst shall first repeat the tests in Section 8.2 to demonstrate that the requirements of
Section 8.2 can be met using the cleanup procedure(s) as an integral part of the method. Figure 1
outlines the cleanup steps.
11.1.1 Gel permeation chromatography (Section 11.2) removes many high molecular weight
interferents that cause GC column performance to degrade. It is used for all soil and
sediment extracts and may be used for water extracts that are expected to contain high
molecular weight organic compounds (e.g., polymeric materials, humic acids).
11.1.2 The solid-phase extraction cartridge (Section 11.3) removes polar organic compounds
such as phenols. It is used for cleanup of organo-chiorine and organo-phosphate extracts.
11.2 Gel permeation chromatography (GPC).
11.2.1 Column packing.
11.2.1.1 Place 70 to 75 g of SX-3 Bio-beads in a 400- to 500-mL beaker.
11.2.1.2 Cover the beads with methylene chloride and allow to swell overnight
(12 hours minimum).
11.2.1.3 Transfer the swelled beads to the column and pump solvent through the
column, from bottom to top, at 4.5 to 5.5 mL/min prior to connecting the
column to the detector.
11.2.1.4 After purging the column with solvent for 1 to 2 hours, adjust the column
head pressure to 7 to 10 psig, and purge for 4 to 5 hours to remove air.
Maintain a head pressure of 7 to 10 psig. Connect the column to the
detector.
11.2.2 Column calibration.
11.2.2.1 Load 5 mL of the calibration solution (Section 6.5) into the sample loop.
11.2.2.2 Inject the calibration solution and record the signal from the detector. The
elution pattern will be corn oil, bis(2-ethylhexyl) phthalate,
pentachiorophenol, perylene, and sulfur.
11.2.2.3 Set the “dump time to allow >85% removal of the corn oil and > 85%
collection of the phthalate.
11.2.2.4 Set the “collect time” to the peak minimum between perylene and sulfur.
11.2.2.5 Verify the calibration with the calibration solution after every 20 extracts.
Calibration is verified if the recovery of the pentachiorophenol is greater than
85%. If calibration is not verified, the system shall be recalibrated using the
699
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Method 1657
calibration solution, and the previous 20 samples shall be re-extracted and
cleaned up using the calibrated GPC system.
11.2.3 Extract cleanup: GPC requires that the column not be overloaded. The column specified
in this method is designed to handle a maximum of 0.5 g of high molecular weight
material in a 5 mL extract. If the extract is known or expected to contain more than
0.5 g, the extract is split into fractions for GPC and the fractions are combined after
elution from the column. The solids content of the extract may be obtained
gravimetrically by evaporating the solvent from a 50- tL aliquot.
11.2.3.1 Filter the extract or load through the filter holder to remove particulates.
Load the 5.0 mL extract onto the column.
11.2.3.2 Elute the extract using the calibration data determined in Section 11.2.2.
Collect the eluate in a clean 400- to 500-mL beaker.
11.2.3.3 Rinse the sample loading tube thoroughly with methylene chloride between
extracts to prepare for the next sample.
11.2.3.4 If a particularly dirty extract is encountered, a 5.0 mL methylene chloride
blank shall be run through the system to check for carry-over.
11.2.3.5 Concentrate the extract and exchange into hexane per Sections 10.5 and 10.6.
Adjust the final volume to 5.0 mL.
11.3 SoLid-phase extraction (SPE).
11.3.1 Setup.
11.3.1.1 Attach the Vac-elute manifold to a water aspirator or vacuum pump with the
trap and gauge installed between the manifold and vacuum source.
11.3.1.2 Place the SPE cartridges in the manifold, turn on the vacuum source, and
adjust the vacuum to 5 to 10 psia.
11.3.2 Cartridge washing: Pre-elute each cartridge prior to use sequentially with 10 mL
portions each of bexane, methanol, and water using vacuum for 30 seconds after each
elnait Follow this pre-elution with 1 mL methylene chloride and three 10 mL portions
of the ehition solvent (Section 6.6.2.2) using vacuum for 5 minutes after each eluant.
Tsp the cartridge lightly while under vacuum to dry between eluants. The three portions
of elution solvent may be collected and used as a blank if desired. Finally, elute the
cartridge with 10 mL each of methanol and water, using the vacuum for 30 seconds after
each eluant.
11.3.3 Cartridge certification: Each cartridge lot must be certified to ensure recovery of the
compounds of interest and removal of 2,4,6-trichiorophenol.
11.3.3.1 To make the test mixture, add the trichlorophenol solution (Section 6.6.2.1)
to the combined calibration standard (Section 7.4). Elute the mixture using
the procedure in Section 11.3.4.
11.3.3.2 Concentratetheeluantto 1.OmL and inject 1.0 &L of the concentrated eluant
into the GC using the procedure in Section 13. The recovery of all analytes
7
-------
Method 1657
(including the unresolved GC peaks) shall be within the ranges for recovery
specified in Table 4, and the peak for trichiorophenol shall not be detectable;
otherwise the SPE cartridge is not performing properly and the cartridge lot
shall be rejected.
11.3.4 Extract cleanup.
1 1.3.4.1 After cartridge washing (Section 11.3.2), release the vacuum and place the
rack containing the 50-mL volumetric flasks (Section 5.6.2.4) in the vacuum
manifold. Reestablish the vacuum at 5 to 10 psia.
11.3.4.2 Using a pipette or a 1-mL syringe, transfer 1.0 mL of extract to the SPE
cartridge. Apply vacuum for 5 minutes to dry the cartridge. Tap gently to
aid in drying.
11.3.4.3 Elute each cartridge into its volumetric flask sequentially with three 10-mL
portions of the elution solvent (Section 6.6.2.2), using vacuum for 5 minutes
after each portion. Collect the eluants in the 50-mL volumetric flasks.
11.3.4.4 Release the vacuum and remove the 50-mL volumetric flasks.
11.3.4.5 Concentrate the eluted extracts to approximately 0.5 mL using the nitrogen
blow-down apparatus. Adjust the final volume to 1.0 mL (per Section 10.6)
and proceed to Section 13 for GC analysis.
12. GAs CHROMATOGRAPHY
Table 2 summarizes the recommended operating conditions for the gas chromatograph. Included in this
table are the retention times and estimated detection limits that can be achieved under these conditions.
Examples of the separations achieved by the primary and confirmatory columns are shown in Figure 2.
12.1 Calibrate the system as described in Section 7.
12.2 Set the autosampler to inject the same volume that was chosen for calibration (Section 7.3.1.3)
for all standards and extracts of blanks and samples.
12.3 Set the data system or GC control to start the temperature program upon sample injection, and
begin data collection after the solvent peak elutes. Set the data system to stop data collection after
the last analyte is expected to elute and to return the column to the initial temperature.
13. SYSTEM AND LABOR4 TORY PERFORMANCE
13.1 At the beginning of each 8-hour shift during which analyses are performed, GC system
performance and calibration are verified for all pollutants and surrogates on both column/detector
systems. For these tests, analysis of the combined QC standard (Section 7.4) shall be used to
verify all performance criteria. Adjustment and/or recalibration (per Section 7) shall be performed
until all performance criteria are met. Only after all performance criteria are met may samples,
blanks, and precision and recovery standards be analyzed.
13.2 Retention times: The absolute retention times of the peak maxima shall be within ±10 seconds
of the retention times in the initial calibration (Section 7.4.1).
701
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Method 1657
13.3 GC resolution: Resolution is acceptable if the valley height between two peaks (as measured from
the baseline) is less than 10% of the taller of the two peaks.
13.3.1 Primary column (DB-1): Malathion and ethyl parathion.
13.3.2 Confirmatory column (DB-1701): Terbufos and diazinon.
13.4 Calibration verification: Calibration is verified for the combined QC standard only.
13.4.1 Inject the combined QC standard (Section 7.4)
13.4.2 Compute the percent recovery of each compound or coeluting compounds, based on the
calibration data (Section 7.4).
13.4.3 For each compound or coeluted compounds, compare this calibration verification
recovery with the corresponding limits for ongoing accuracy in Table 4. For coeluting
compounds, use the coeluted compound with the least restrictive specification (the widest
range). If the recoveries for all compounds meet the acceptance criteria, system
performance is acceptable and analysis of blanks and samples may begin. If, however,
any recovery falls outside the calibration verification range, system performance is
unacceptable for that compound. In this case, correct the problem and repeat the test,
or recalibrate (Section 7).
13.5 Ongoing precision and recovery.
13.5.1 Analyze the extract of the precision and recovery standard extracted with each sample
batch (Sections 10.2.3.3 and 10.2.5.7).
13.5 2 Compute the percent recovery of each analyte and coeluting compounds.
13.5.3 For each compound or coeluted compounds, compare the percent recovery with the limits
for ongoing recovery in Table 4. For coeluted compounds, use the coeluted compound
with the least restrictive specification (widest range). If all analytes pass, the extraction,
concentration, and cleanup processes arc in control and analysis of blanks and samples
may proceed. If, however, any of the analytes fail, these processes are not in control.
In this event, correct the problem, re-extract the sample lot, and repeat the ongoing
precision and recovery test
13.5.4 Add results which pass the specifications in Section 13.6.3 to initial and previous ongoing
data. Update QC charts to form a graphic representation of continued laboratory
performance. Develop a statement of laboratory data quality for each analyte by
caJcuI ing the average peicent recovery (R) and the standard deviation of percent
recovery a,. Express the accuracy as a recovery interval from R - 2S to R + 2Sr. For
eiample,ifR=95%ands=5%,theaccuracyis85to l05%.
14. QuAliTATIvE DETER NA TION
14.1 Qualitative determination is accomplished by comparison of data from analysis of a sample or
blank with data from analysis of the shift standard (Section 14.2), and with data stored in the
retention-time and calibration libraries (Sections 7.3.2 and 7.3.3.2). Identification is confirmed
when retention time and amounts agree per the criteria below.
702
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Method 1657
14.2 For each compound on each column/detector system, establish a retention-time window ± 20
seconds on either side of the retention time in the calibration data (Section 7.3.2). For compounds
that have a retention-time curve (Section 7.3.2.2), establish this window as the minimum
-20 seconds and maximum +20 seconds.
14.2.1 Compounds not requiring a retention-time calibration curve: If a peak from the analysis
of a sample or blank is within a window (as defined in Section 14.2) on the primary
column/detector system, it is considered tentatively identified. A tentatively identified
compound is confirmed when (1) the retention time for the compound on the
confirmatory column/detector system is within the retention-time window on that system,
and (2) the computed amounts (Section 16) on each system (primary and confirmatory)
agree within a factor of 3.
14.2.2 Compounds requiring a retention-time calibration curve: If a peak from the analysis of
a sample or blank is within a window (as defined in Section 14.2) on the primary
column/detector system, it is considered tentatively identified. A tentatively identified
compound is confirmed when (1) the retention times on both systems (primary and
confirmatory) are within ±30 seconds of the retention times for the computed amounts
(Section 15), as determined by the retention-time calibration curve (Section 7.3.2.2), and
(2) the computed amounts (Section 15) on each system (primary and confirmatory) agree
within a factor of 3.
15. QUANTITATIVE DETERMINA TION
15.1 Using the GC data system, compute the concentration of the analyte detected in the extract (in
micrograms per milliliter) using the calibration factor or calibration curve (Section 7.3.3.2).
15.2 Liquid samples: Compute the concentration in the sample using the following equation:
Equation 4
cs=10 :
where
C, = The concentration in the sample, in gfL
10 = The extract total, in mL
= The concentration in the extract, in j g/mL
V 3 = The sample extracted, in L
703
-------
Method 1657
15.3 Solid samples: Compute the concentration in the solid phase of the sample using the following
equation:
Equation 5
c=1o
1 1000(W,)(solids)
where
C, = Concentration In the sample, in ,iglkg
10 = Extract total, in mL
C, = Concentration in the extract, in g/mL
1000 = Conversion factor, g to kg
= Sample weight, in g
solids = Percent solids in Section 10.1.3 divided by 100
15.4 If the concentration of any analyte exceeds the calibration range of the system, the extract is
diluted by a factor of 10, and a 1 -FL aliquot of the diluted extract is analyzed.
15.5 Report results for all pollutants found in all standards, blanks, and samples to three significant
figures. Results for samples that have been diluted are reported at the least dilute level at which
the concentration is in the calibration range.
16. ANALYSIS OF COMPLEX SAMPLES
16.1 Seine samples may contain high levels (>1000 ng/L) of the compounds of interest, interfering
compounds, and/or polymeric materials. Some samples may not concentrate to 10 mL
(Section 10.6); others may overload the GC column and/or detector.
16.2 The analyst shall attempt to clean up all samples using GPC (Section 11.2), and the SPE cartridge
(Section 11.3). If these techniques do not remove the interfering compounds, the extract is diluted
by a factor of 10 and reanalyzed (Section 16.4).
16.3 Recoveçy of surrogates: In most samples, surrogate recoveries will be similar to those from
reagent water or from the high solids reference matrix. If the surrogate recovery is outside the
range specified in Section 8.3, the sample shall be re-extracted and reanalyzed. If the surrogate
recovery is still outside this range, the sample is diluted by a factor of 10 and reanalyzed
(Section 15.4).
16.4 Recovery of matrix spikes: In most samples, matrix spike recoveries will be similar to those from
reagent water or from the high solids reference matrix. If the matrix spike recovery is outside
the range specified in Table 4, the sample shall be diluted by a factor of 10, respited, and
reanalyzed. If the matrix spike recovery is still outside the range, the method may not apply to
the sample being analyzed and the result may not be reported for regulatory compliance purposes.
17. hI ff00 PERFORMANCE
17.1 Development of this method is detailed in References 8 and 9.
704
-------
Method 1657
References
1. “Carcinogens: Working with Carcinogens.” Department of Health, Education, and Welfare;
Public Health Service; Center for Disease Control; National Institute for Occupational Health and
Safety: Publication 77-206, Aug 1977.
2. “OSHA Safety and Health Standards, General Industry” (29 CFR 1910). Occupational Safety and
Health Administration: Jan 1976.
3. “Safety in Academic Chemistry Laboratories,” American Chemical Society Committee on
Chemical Safety: 1979.
4. Mills, P. A., “Variation of Florisil Activity: Simple Method for Measuring Adsorbent Capacity
and Its Use in Standardizing Florisil Columns,” Journal of the Association of Official Analytical
Chemists, 51, 29: 1968.
5. “Handbook of Quality Control in Wastewater Laboratories,” U.S. Environmental Protection
Agency, Environmental Monitoring and Support Laboratory, Cincinnati, OH,: EPA-600/4-79 -019,
March 1979.
6. “Standard Practice for Sampling Water” (ASTM Annual Book of Standards), American Society
for Testing and Materials, Philadelphia, Pennsylvania: 76, 1980.
7. “Methods 330.4 and 330.5 for Total Residual Chlorine,” U.S. Environmental Protection Agency,
Environmental Monitoring and Support Laboratory, Cincinnati, OH: EPA-600/4-70-020, March
1979.
8. “Consolidated GC Method for the Determination of ITDIRCRA Pesticides using Selective GC
Detectors,” S-CUBED, A Division of Maxwell Laboratories, Inc., La Jolla, CA: Ref. 32145-01,
Document RiO, September 1986.
9. “Method Development and Validation, EPA Method 1618,” Pesticide Center, Department of
Environmental Health, Colorado State University: November 1988, January 1989, and
March 1992.
705
-------
Method 1657
Table 1. Organo-Phosphorus Pesticides Determined by Large-Bore, Fused-Silica
Capillary Column Gas Chromatography with Flame Photometric
Detector
EPA
EGD Compowid CAS Registry
Acephate 30560-19-1
468 Azinphos ethyl 2642-71-9
453 Azinphos methyl 86-50-0
461 Chlorfevinphos 470-90-6
469 Cblorpyrifos 2921-88-2
443 Coumaphos 56-72-4
479 Crotoxyphos 7700-17-6
DEF 78-48-8
471 Demeton 8065-48-3
460 Diazmon 333-41-5
Dicb lofenthion 97-17-6
450 Diddorvos 62-73-7
455 Dicrotophos 141-66-2
449 Ditnethoate 60-51-5
452 Diozathion 78-34-2
458 Disulfoton 298-04-4
467 EPN 2104-64-5
463 Ethion 563-12-2
Ethoprop 13194-48-4
446 Famphur 52-85-7
454 Fensulfothion 115-90-2
447 Fe”d’ioa 55-38-9
464 Hexamethylphospnoramioe 680-31-9
474 Leptophos 21609-90-5
475 Malathion 12 1-75-5
Merphos 150-50-5
Methamidophos 10265-92-6
Methyl chlorpyrifos 5598-13-0
456 Methyl parathion 298-00-0
Methyl trithion 953-17-3
444 Mevinphos 7786-34-7
470 Moaocrotophos 6923-22-4
459 Naled 300-76-5
448 Parathion (ethyl) 56-38-2
457 Thorate 298-02-2
465 Phosinet 732-11-6
473 Phosphainidon 13171-21-6
Ronnel 299-84-3
706
-------
Method 1657
Table 1. Organo-Phosphorus Pesticides Determined by Large-Bore, Fused-Silica
Capillary Column Gas Chromatography with Flame Photometric
Detector (cant.)
EPA
EGD Compound CA S Registry
477 Sulfotepp 3689-24-5
Suiprofos (Bolstar) 35400-43-2
476 TEPP 107-49-3
472 Terbufos 1307 1-79-9
466 Tetrachlorvinphos 961-11-5
Tokutbion 34643-464
445 Trichiorfon 52-68-6
Trichloronate 327-98-0
451 Tricresylphosphate 78-30-8
462 Trimethylphosphate 512-56-1
707
-------
Method 1657
Table 2. Gas Chromatography of Organo-Phosphorus Pesticides
Retention Time 1
(mini
EPA Compowid DB- 1 OH- 1701 MDL 2
EGO (ng/Li
450 Dichlorvos 6.56 9.22 4
444 Mevinphos 11.85 16.20 74
Acephate 12.60 17.40 500
445 Trich1oro1 rn 12.69 18.85 150
Methaznidophos 15.10 19.20 100
471 Demeton-A 17.70 20.57 19
Ethoprop 18.49 21.43 7
459 Naled 18.92 23.00 18
455 Dicrotophos 19.33 26.30 81
470 Monocrotophos 19.62 29.24 85
477 Sulfotepp 20.04 23.68 6
457 Phorate 20.12 23.08 10
449 Dimethoate 20.59 29.29 27
Demeton-B 21.40 25.52 21
452 Dioxathion 22.24 26.70 121
472 Terbufos 22.97 24.55 26
473 Phosphamidon-E 23.70 29.89 28
458 Disulfoton 23.89 27.01 32
460 Diazinon 24.03 26.10 38
Trlbutyl phosphate (surr) 24.50 17.20 -
Pbosphamidon-Z 25.88 32.62 116
456 Methyl parathion 25.98 32.12 18
Dicblorofenthion 26.11 28.66 6
Methyl chlorpyrilbs 26.29 29.53 13
Ronnel 27.33 30.09 11
475 Malathion 28.87 33.49 11
447 Fenthion 29.14 32.16 22
448 P&uthion 29.29 34.61 10
469 Chiorpyrlfbs 29.48 32.15 4
Trichloronate 30.44 32.12 14
461 Chlorfevinphos 32.05 36.08 2
479 Crotoxyphos 32.65 37.58 81
Tokutluon 33.30 37.17 2
466 T achlorvinphos 33.40 37.85 12
DEF 34.05 37.50 50
Merphos-B 35.16 37.37 18
454 Fensulfothion 36.58 43.86 104
Methyl trithion 36.62 40.52 10
463 Ethion 37.61 41.67 13
708
-------
Method 1657
Table 2. Gas Chromatography of Organo-Phosphorus Pesticides (cont.)
Retention Time 1
(mini
EPA Compound DB- 1 OB- 1701 MDL 2
EGO (ng/L)
Sulprofos 38.10 41.74 6
446 Famphur 38.24 46.37 27
465 Phosmet 41.24 48.22 14
467 EPN 41.94 47.52 9
453 Azinphos methyl 43.33 50.26 9
474 Leptophos 44.32 47.36 14
468 Azinphos ethyl 45.55 51.88 22
Triphenyl phosphate (surr) 47.68 40.43 -
443 Coumaphos 48.02 56.44 24
Notes:
1. Columns: 30 m long by 0.53 mm ID; DB-1: 1.5 &; DB-1701: 1.0 , . Conditions suggested to meet
retention times shown: 110°C for 0.5 mm, 110 to 250° at 3°C/mm, 250°C until coumaphos elutes.
Carrier gas flow rate approximately 7 mL/min.
2. 40 CFR Part 136, Appendix B (49 FR 43234).
3. Estimated. Detection limits for soils (in ng/kg) are estimated to be 30 to 100 times this level.
709
-------
Method 7657
Table 3. Concentrations of Calibration Solutions
Concentration(pg/mL)
Compowid Low Medium High
Calibration group 1
453 Azinphos methyl 0.1 0.5 2.0
450 Dichlorvos 0.5 2.5 10.0
458 Disulfbton 0.2 1.0 4.0
447 Fenthion 0.2 1.0 4.0
M phos-A 0.2 1.0 4.0
Merphos-B 0.2 1.0 4.0
Methyl trithion 0.5 2.5 10.0
Ronnel 0.2 1.0 4.0
Suiprolbs 0.2 1.0 4.0
Calibration group 2
461 ailorfevinphos 0.2 1.0 4.0
469 Qilorpyrilbs 0.2 1.0 4.0
471 Demeton-A 0.2 1.0 4.0
Dem e ton-B 0.2 1.0 4.0
Dichlolènthion 0.2 1.0 4.0
449 Dimethoate 0.1 0.5 2.0
446 Famphur 0.5 2.5 10.0
474 Lóptoçhos 0.2 1.0 4.0
456 Methyl parathion 0.2 1.0 4.0
445 Trlchlorolbn 0.5 2.5 10.0
451 Tricresylphosphate 1.0 5 20.0
Ca ration group 3
468 Aziojthos ethyl 0.2 1.0 4.0
479 Crotoayphos 0.5 2.5 10.00
DEF 0.2 1.0 4.0
454 FeuLsullbthion 0.5 2.5 10.0
Methyl chlorpyrifos 0.2 1.0 4.0
444 Mevinphos 0.5 2.5 10.0
459 NalecI 0.5 2.5 10.0
448 Parathion 0.2 1.0 4.0
465 Pbosmet 0.5 2.5 10.0
473 Pliospbamldon-E 0.5 2.5 10.0
Phosphainidon-Z 0.5 2.5 10.0
477 Sul1btq ip 0.2 1.0 4.0
472 T buks 0.2 1.0 4.0
ca aauon soup 4
443 Coumaphos 0.5 2.5 10.0
460 Disnnon 0.2 1.0 4.0
467 EPN 0.2 1.0 4.0
710
-------
Method 1657
Table 3. Concentrations of Calibration Solutions (cont.)
Concentration(pg/mL)
EPA
EGD Compound Low Medium High
463 Ethion 0.2 1.0 4.0
Ethoprop 0.2 1.0 4.0
475 Malathion 0.2 1.0 4.0
457 Phorate 0.2 1.0 4.0
466 Tetrachlorvinphos 0.2 1.0 4.0
Trichloronate 0.2 1.0 4.0
711
-------
Method 1657
Table 4. Acceptance Criteria for Performance Tests for Organo-Phosphorus
Compounds
Acceptance Criteria
EGO
No.
initial Precision
and
Calibration
Recovery
Spike
Accuracy
Verification
Ongoing
Level
(%)
Accuracy
Compoimd
(ng/L)
s’
x
(pg/mU
R (%)
Acephate
50000
25 32
- 122
68 - 132
28 - 126
468
Azinphos ethyl
200
10 71
-117
77 - 127
59 -129
453
Azinphos methyl
100
10 52
- 112
83 - 119
37 - 127
461
Ch1orf vinphos
200
11 56
- 132
83 - 114
37 - 151
469
Chkirpyrifos
200
10 61
- 112
80 - 119
48 - 125
443
Coumaphos
500
10 78
- 104
82 - 120
72 -‘110
479
Crotoxyphos
500
46 28
- 116
68 - 136
6 - 138
DEF
1500
31 45
- 107
68 - 132
•
42 - 110
471
Demeton
200
23 33
- 101
64 - 123
16 - 118
460
Diazh n
Didilofenthion
Dichlorvos
200
200
500
10 70
10 75
18
- 110
- 115
52-106
86 - 114
80 - 110
77- 103
60 - 120
65 - 125
39-119
450
455
Dicn*ophos
not recovered
78 - 122
449
Dimethoate
100
89 27
- 100
73 - 127
22 - 100
452
Dmxath lon
600
22 59
- 101
79 - 121
49 - 111
458
Disulfoton
200
30 46
- 98
70 - 118
33 - 111
467
EPN
200
13 74
- 124
81 - 108
62 - 136
463
Ethion
200
11 72
- 134
70 - 118
47 - 149
Ethoprop
200
14 79
- 103
84 - 108
73 - 109
446
Famphur
500
12 81
- 101
81 - 113
76 - 106
454
F Ifi*hion
500
65
13-115
42- 139
0-141
447
464
Fenthion
200
13 69
not recovered
- 101
73 - 137
70 - 130
61 - 109
Hexamethytphos-
—
474
Leptophos
200
10 85
- 105
85 - 112
80 - 110
475
Malathion
200
10
75-109
82- 108
66-118
Meq,hos-B
Methamidophos’
Methylchlorpyrifbs
200
10000
200
10
33
10 88
68-102
66-132
-108
72- 118
70- 128
81 - 114
59-111
63-135
83 -113
456
Methylparathion
200
15
72-112
89- 114
61 -123
Methyltrithlon
500
20
21-137
78- 122
0-166
444
470
459
Mev Ii*os
Monocrotoiihos
Naled
500
500
23
not recovered
10 0
24-100
- 148
73- 135
19 - 206
77 - 114
7-107
0 - 176
448
Par liion
200
10
71-111
79- 110
61 -121
457
Phorate ‘
200
19 54
- 100
70 - 118
43 - 109
465
lOsiflet
500
39
44-119
61- 159
25-138
772
-------
Method 1657
Table 4. Acceptance Criteria for Performance Tests for Organo-Phosphorus
Compounds (cant.)
Notes:
‘With salt and azeotropic extraction
2 With salt
Acceptance Criteria
EGO
No.
Spike
Level
(ng/L)
Initial Precision and
Accuracy
(%1
Calibration
Verification
Compound
s
x
(pg/mL)
473
Phosphamidon-Z
500
45
0 - 100
81 -
102
Ronnel
200
10
79 - 111
78 -
113
477
Sultbtepp
200
10
70 - 120
75 - 115
Su lprofos
200
10
75 - 100
81 - 118
476
TEPP
not recovered
70 - 130
472
Terbufos
200
23
60 - 110
82 - 111
466
Tetrachlorvinphos
200
11
48 - 110
73 - 119
Tokuthion
100
17
73 - 105
70 - 130
445
Trichlorofon 2
50000
42
43 - 195
58 - 142
Trichloronate
200
10
82 - 102
80 - 113
451
Tricresylphosphate
1000
10
81 - 101
70 - 130
462
Trimethylphosphate
not recovered
70 - 130
Recovery
Ongoing
Accuracy
R(%)
0 - 100
71 -119
58 - 132
70 - 100
47 - 123
32-126
65 -113
37 -201
77 - 107
74 - 114
71 .
-------
Method 1657
I
[ PercerdSohdsl
Methan*lophos Noimar Coiiounds
I
IM t
I
I CHCl 3 :(CH J 2 CO IL Ext ]
I
To Cleanup
Extraction arid Conceidration Steps
From Extraction
I G&Penneatbn I
I
I Soid PhaseExt1
<30% SolIds
I
I
> 30% Solids
I
ACN & CH 2 C1 2 Sonic
CH 2 CI 2 Uq.Llq. Ext
H 2 0 Back Extract
I I
[ c o n c e ej
I
GC/FPD
Cleaiijp and Analysis Steps
Figure 1. Extraction, Cleanup, Derivatization, and Analysis
714
-------
Method 1857
0
U
I
C
0
0
0
0
U
C
2 0
2 .
E e
LI
C 61
o
0
61
C
0
‘C
C
E
61
C
0
‘ C
610
0
a
jI [
C
0
‘C
61
61
-9
‘ C
61
a
0
0
a
‘C
2
a
I 0
I
F 2 I 2
61
JLI LI.
z 2
0. a C
w
61
‘5.
61
E
.4k
0
‘C
0
— .0 61
2
Z ‘5.
61 E
W ‘5. D
E 0
U
L - .
6 7 8 9 10 11 12 13 15 5 i7 18 19 20 21 22 23 24 2526 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 4 48 49 50 1 52 53 54 1 56
Retention Time (minutes)
A -OO2 .88
Figure 2. Gas Chromatogram of Selected Organo-Phosphorus Compounds.
(B)
(A)
0
0
a
715
-------
Method 1658
The Determination of
Phenoxy -Acid Herbicides in
Municipal and Industrial
Waste water
-------
Method 1658
The Determination of Phenoxy-Acid Herbicides in Municipal and
Industrial Wastewater
SCOPE AND APPLICA T1ON
1.1 This method is designed to meet the survey requirements of the Environmental Protection Agency
(EPA). It is used to determine (I) the phenoxy-acid herbicides and herbicide esters associated
with the Clean Water Act, the Resource Conservation and Recovery Act, and the Comprehensive
Environmental Response, Compensation and Liability Act; and (2) other compounds amenable to
extraction and analysis by automated, wide-bore capillary column gas chromatography (GC) with
electron capture or halogen-selective detectors.
1.2 The chemical compounds listed in Table I may be determined in waters, soils, sediments, and
sludges by this method. This method should be applicable to other herbicides. The quality
assurance/quality control requirements in this method give the steps necessary to determine this
applicability.
1.3 When this method is applied to analysis of unfamiliar samples, compound identity must be
supported by at least one additional qualitative technique. This method describes analytical
conditions for a second gas chromatographic column that can be used to confirm measurements
made with the primary column. Gas chromatography mass spectrometry (GC/MS) can be used
to confirm compounds in extracts produced by this method when analyte levels are sufficient.
1.4 The detection limit of this method is usually dependent on the level of interferences rather than
instrumental limitations. The limits in Table 2 typify the minimum quantity that can be detected
with no interferences present.
1.5 This method is for use by or under the supervision of analysts experienced in the use of a gas
chromatograph and in the interpretation of gas chromatographic data. Each laboratory that uses
this method must demonstrate the ability to generate acceptable results using the procedure in
Section 8.2.
2. SUMMARY OF METHOD
2.1 Extraction.
2.1.1 The percent solids content of a sample is detennined.
2.1.2 Samples containing low solids: If the solids content is less than or equal to 1%, the
sample is extracted directly using continuous extraction techniques. The pH of a 1-L
sample raised to 12 to 13 to hydrolyze acid esters, and the sample is extracted with
methylene chloride to remove interferences. The pH is lowered to less than 2 and the
free acids are extracted with methylene chloride.
719
-------
Method 1658
2.1.3 Samples containing greater than I % sohds.
2.1.3.1 Solids content I to 30%: The sample is diluted to I % solids with reagent
water, homogenized ultrasonicaHy, and extracted as a low-solids sample
(Section 2.1.2).
2.1.3.2 Solids content greater than 30%: The sample is placed in an extraction bottle
and approximately 1-L of basic (pH 12-13) water is added. The bottle is
tumbled for 18 hours. The water is removed and extracted as a tow-solids
sample (Section 2.1.2).
2.2 Concentration and cleanup: The extract is dried over sodium sulfate, concentrated using a
Kuderna-Danish evaporator, cleaned up (if necessary) using gel permeation chromatography
(GPC) and concentrated to 5 or 10 mL (depending upon whether GPC was or was not used).
2.3 Derivatization and cleanup: The acids in the extract are derivatized to form the methyl esters.
The solution containing the methyl esters is cleaned up (if necessary) using solid-phase extraction
(SPE) and/or adsorption chromatography and reconcentrated to 5 or 10 mL.
2.4 Gas chromatography: A 1- 1 L aliquot of the extract is injected into the gas chromatograph (GC).
The dótivatized acids are separated on a wide-bore, fused-silica capillary column and are detected
by an electron capture, microcoulometric, or electrolytic conductivity detector.
2.5 Identification of a pollutant (qualitative analysis) is performed by comparing the GC retention
limes of the compound on two dissimilar columns with the respective retention times of an
authentic standard. Compound identity is confirmed when the retention times agree within their
— windows.
2.6 Quantitative analysis is performed by using an authentic standard to produce a calibration factor
or calibration curve, and using the calibration data to determine the concentration of a pollutant
in the extract. The concentration in the sample is calculated using the sample weight or volume
and the extract volume.
2.7 Quality is assured through reproducible calibration and testing of the extraction and GC systems.
3. CONTAJINATION AND INTERFERENCES
3.1 Solvents, reagents, glassware, and other sample-processing hardware may yield artifacts and/or
elevated baselines causing misinterpretation of chromatograms. All materials used in the analysis
shall be demonstrated to be free from interferences under the conditions of analysis by running
method bIank as described in Section 8.5.
3.2 Glassware and, where possible, reagents are cleaned by solvent rinse and baking at 450°C for
1 hour mininiun in a muffle furnace or kiln. Some thermally stable materials, such as PCBs, may
not be eIimin ad by this treatment and thorough rinsing with acetone and pesticide-quality hexane
may be required.
3.3 SpecifIc selection of reagents and purification of solvents by distillation in all-glass systems may
be required.
3.4 Intethrence by phthalate esters can pose a major problem in herbicide analysis when using the
electron capture detector. Phthalates usually appear in the chromatograin as large, late-eluting
peaks. Phthalates may be leached from common flexible plastic tubing and other plastic materials
during the extraction and clean-up processes. Cross-contamination of clean glassware routinely
720
-------
Method 1658
occurs when plastics are handled during extraction, especially when solvent-wetted surfaces are
handled. Interferences from phthalates can best be minimized by avoiding the use of plastics in
the laboratory, or by using a microcoulometric or electrolytic conductivity detector.
3.5 The acid forms of the herbicides are strong acids that react readily with alkaline substances and
can be lost during analysis. Glassware, glass wool, and all other apparatuses should be rinsed with
dilute hydrochloric or sulfuric acid prior to use. Sodium sulfate and other reagents that can be
acidified should be acidified to preclude the herbicides from being adsorbed by these reagents.
3.6 Organic acids and phenols cause the most direct interference with the herbicides. Alkaline
hydrolysis and subsequent extraction of the basic solution can remove many hydrocarbons and
esters that may interfere with the herbicide analysis.
3.7 Interferences coextracted from samples will vary considerably from source to source, depending
on the diversity of the site being sampled. The cleanup procedures given in this method can be
used to overcome many of these interferences, but unique samples may require additional cleanup.
to achieve the minimum levels given in Table 2.
4. SAFETY
4.1 The toxicity or carcinogenicity of each compound or reagent used in this method has not been
precisely determined; however, each chemical compound should be treated as a potential health
hazard. Exposure to these compounds should be reduced to the lowest possible level. The
laboratory is responsible for maintaining a current awareness file of OSHA regulations regarding
the fe handling of the chemicals specified in this method. A reference file of material handling
sheets should also be made available to all personnel involved in these analyses. Additional
information on laboratory safety can be found in References 1 through 3.
4.2 Primary standards of hazardous compounds shall be prepared in a hood, and a NIOSH/MESA
approved toxic gas respirator should be worn when high concentrations are handled.
4.3 Diazomethane is a toxic carcinogen which can decompose or explode under certain conditions.
Solutions decompose rapidly in the presence of solid materials such as copper powder, calcium
chloride, and boiling chips. The following operations may cause explosion: heating above 90°C;
use of grinding surfaces such as ground-glass joints, sleeve bearings, and glass stirrers; and
storage near alkali metals. Diazomethane shall be used only behind a safety screen in a well
ventilated hood and should be pipetted with mechanical devices only.
4.4 Unknown samples may contain high concentrations of volatile toxic compounds. Sample
containers should be opened in a hood and handled with gloves that will prevent exposure. The
oven used for sample drying to determine percent moisture should be located in a hood so that
vapors from samples do not create a health hazard in the laboratory.
5. APPARA TUS AND MA TERIALS
NOTE: Brand names, suppliers, and part numbers are for illustrative purposes only. No
endorsement is implied. Equivalent performance may be achieved using apparatus and
materials other than those spec ed here, but de,nonstration of equivalent performance
meeting requirements of this method is the responsibility of the laboratory.
721
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Method 1658
5.1 Sampling equipment for discrete or composite sampling.
5.1.1 Sample bottles and caps.
5.1.1.1 Liquid samples (waters, sludges and similar materials that contain less
than 5% solids): Sample bottle, amber glass, 1-L or 1-quart, with screw-cap.
5.1.1.2 Solid samples (soils, sediments, sludges, filter cake, compost, and similar
materials that contain more than 5% solids): Sample bottle, wide-mouth,
amber glass, 500-mL minimum.
5.1.1.3 If amber bottles are iK* available, samples shall be protected from light.
5.1.1.4 Bottle caps: Threaded to fit sample bottles. Caps shall be lined with PTFE.
5.1.1.5 aeaning.
5.1.1.5.1 Bottles are detergent-water washed, then rinsed with solvent
rinsed or baked at 450°C for 1 hour minimum before use.
5.1.1.5.2 Liners are detergent-water washed, then rinsed with reagent water
and solvent, and baked at approximately 200°C for 1 hour
minimum prior to use.
5.1.2 Compositizig equipment: Automatic or manual compositing system incorporating glass
containers cleaned per bottle cleaning procedure above. Sample containers are kept
at 0 to 4°C during sampling. Glass or 1 FFE tubing only shall be used. If the sampler
uses a peristaltic pump, a minimum length of compressible silicone rubber tubing may
be used in the pump only. Before use, the tubing shall be thoroughly rinsed with
methanol, followed by repeated rinsings with reagent water to minimize sample
contamination. An Integrating flow meter is used to collect proportional composite
5.2 Equipment for determinIng percent moisture.
5.2.1 Oven, capable of maint3ining a temperature of 110°C (±5°C).
5.2.2 Dessicator.
5.2.3 Crucibles, porcelain.
5.2.4 Weiglibig pme, aluminum .
5.3 Extraction equipment.
5.3.1 Equipment fOr ultrasonic extraction.
5.3.1.1 Sonic disruptor 375 watt with pulsing capability and ½- or %-inch.
disruptor horn (Ultrasonics, mc, Model 375C, or equivalent).
5.3.1.2 Sonabox (or equivalent), for use with disruptor.
5.3.2 Equipment for liquid-liquid extraction.
5.3.2.1 Contimious liquidiquid extractor: PTFE or glass connecting joints and
stopcocks without lubrication, 1.5- to 2-L (Hershberg-Wolf Extractor,
Cal-Glass, Costa Mesa,California,. 1000- or 2000-mL continuous extractor,
or equivalent).
5.3.2.2 Round-bottom flask, S00-mL, with heating mantle.
722
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Method 1658
5.3.2.3 Condenser, Graham, to fit extractor.
5.3.2.4 pH meter, with combination glass electrode.
5.3.2.5 pH paper, wide range (Hydrion Papers, or equivalent).
5.3.3 Separatory funnels: 250-, 500-, 1000-, and 2000-mL, with PTFE stopcocks.
5.3.4 Filtration apparatus.
5.3.4.1 Glass powder funnels: 125- to 250-mL.
5.3.4.2 Filter paper for above (Whatman 41, or equivalent).
5.3.5 Beakers.
5.3.5.1 1.5- to 2-L, calibrated to I L.
5.3.5.2 400- to 500-mL.
5.3.6 Spatulas: Stainless steel or PTFE.
5.3.7 Drying column: 400 mm long by 15 to 20 mm ID, Pyrex chromatographic column
equipped with coarse glass fit or glass wool plug.
5.3.7.1 Pyrex glass wool: Extracted with solvent or baked at 450°C for 1 hour
minimum.
5.3.8 TLCP extractor.
5.3.8.1 Rotary agitation apparatus: Capable of rotating the extraction vessel in an
end over end fashion at 30 rpm (±2 rpm) (Associated Design and
Manufacturing Co. or equivalent).
5.3.8.2 Bottle, polyethylene or polypropylene, 1- to 4-L, with screw-cap with
PTFE-lined lid, to fit extractor.
5.4 Evaporation/concentration apparatus.
5.4.1 Kuderna-Danish (K-D) apparatus.
5.4.1.1 Evaporation flask: 500-mL (Kontes K-570001-0500, or equivalent), attached
to concentrator tube with springs (Kontes K-662750-0012).
5.4.1.2 Concentrator tube: 10-mL, graduated (Kontes K-570050-1025, or equivalent)
with calibration verified. Ground-glass stopper (size 19/22 joint) is used to
prevent evaporation of extracts.
5.4.1.3 Snyder colunrn: Three-bail macro (Kontes K-503000-0232, or equivalent).
5.4.1.4 Snyder column: Two-ball micro (Kontes K-469002-0219, or equivalent).
5.4.1.5 Boiling chips.
5.4.1.5.1 Glass or silicon carbide: Approximately 10/40 mesh, extracted
with methylene chloride and baked at 450°C for 1 hour
minimum.
5.4.1.5.2 PTFE (optional): Extracted with methylene chloride.
5.4.2 Water bath: Heated, with concentric ring cover, capable of temperature control (±2°C),
installed in a fume hood.
723
-------
Method 1658
5.4.3 Nitrogen evaporation device: Equipped with heated bath that can be maintained at 35 to
40°C (N-Evap, Organomation Associates, Inc., or equivalent).
5.4.4 Sample vials: Amber glass, 1- to 5-mL with PTFE-lined screw- or crimp-cap, to fit GC
auto-sampler.
5.5 Balances.
5.5.1 Analytical: Capable of weighing 0.1 mg.
5.5.2 Top loading: Capable of weighing 10 mg.
5.6 Apparatus for sample cleanup.
5.6.1 Automated gel permeation chroinatograph (Analytical Biochemical Labs, mc, Columbia,
MO, Model GPC Autoprep 1002, or equivalent).
5.6.1.1 Column: 600 to 700 mm long by 25 mm ID, packed with 70 g of SX-3
Blo-beads (Bio-Rad Laboratories, Richmond, CA, or equivalent).
5.6.1.2 Syringe, 10-inL, with Luer fitting.
5.6.1.3 Syringe-filter holder, stainless steel, and glass fiber or PTFE filters (Gelman
ACTOdISC-CR, 1 to 5 micron, or equivalent).
5.6.1.4 UV detectors: 254-mu, preparative or semi-prep flow cell: (Isco, Inc.,
Type 6; Schmadzu, 5 mm path length; Beckman-Altex 152W, 8-pL
micro-prep flow cell, 2-mm path; Pharmacia UV-1, 3-mm flow cell; LDC
Milton-Roy UV-3, monitor #1203; or equivalent).
5.6.2 Vacuum system and cartridges for solid phase extraction (SPE).
5.6.2.1 Vacuum system: Capable of achieving 0.1 bar (house vacuum, vacuum
pump, or water aspirator), with vacuum gauge.
5.6.2.2 VacElute Manifold (Analytichem International, or equivalent).
5.6.2.3 Vacuum trap: Made from 500-mL sidearm flask fitted with single-hole
rubber stopper and glass tubing.
5.6.2.4 Rack for holding 50-mL volumetric flasks in the manifold.
5.6.2.5 Column: Mega Bond Elut, Non-polar, C18 Octadecyl, 10 g/60 mL
(Analytichem International Cat. No.60711060, or equivalent).
5.6.3 Chromatographic column: 400 mm long by 22 mm ID, with I FFE stopcock and coarse
Mt (IContes 1C-42054, or equivalent).
5.7 Crifuge apparatus.
5.7.1 Centrifuge: Capable of rotating 500-mL centrifuge bottles or 15-mi centrifuge tubes at
5,000 rpm minimwn .
5.7.2 Centrifuge bottles: 500-mL, with screw-caps, to fit centrifuge.
5.7.3 Centrifuge tubes: 12- to 15-mL, with screw-caps, to fit centrifuge.
5.7.4 Funnel, Budiner, 15 cm.
5.7.4.1 flask, filter, for use with Buchner funnel.
5.7.4.2 Filter paper, 15 cm (Whatman #41, or equivalent).
724
-------
Method 1658
5.8 Derivatizatlon apparatus: Diazald kit with clear seal joints for generation of diazomethane
(Aldrich Chemical Co. Z1O,025-0, or equivalent).
5.9 MIscellaneous glassware.
Pipettes, glass, volumetric, 1.00-, 5.00-, and 10.0-niL.
Syringes, glass, with Luerlok tip, 0.1-, 1.0- and 5.0-niL. NeedLes for syringes, 2-inch,
22-gauge.
Volumetric flasks, 10.0-, 25.0-, and 50.0-niL.
Scintillation vials, glass, 20- to 50-niL, with PTFE-Iined screw-caps.
5.10 Gas chromatograph: Shall have splitless or on-column simultaneous automated injection into
separate capillary columns with an electron capture or halide-specific detector at the end of each
column, temperature program with isothermal holds, data system capable of recording
simultaneous signals from the two detectors, and shall meet all of the performance specifications
in Section 14.
5.10.1 GC columns: Bonded-phase fused-silica capillary.
5.10.1.1 Primary: 30 m (±3 m) long by 0.5 mm (±0.05 mm) ID (DB-608, or
equivalent).
5.10.1.2 Confirmatory: DB-1701, or equivalent, with same dimensions as primary
column.
5.10.2 Data system shall collect and record GC data, store GC runs on magnetic disk or tape,
process GC data, compute peak areas, store calibration data including retention times and
calibration factors, identify GC peaks through retention times, compute concentrations,
and generate reports.
5.10.2.1 Data acquisition: GC data shall be collected continuously throughout the
analysis and stored on a mass storage device.
5.10.2.2 Calibration factors and calibration curves: The data system shall be used to
record and maintain lists of calibration factors, and multi-point calibration
curves (Section 7). Computations of relative standard deviation (coefficient
of variation) are used for testing calibration linearity. Statistics on initial
(Section 8.2) and ongoing (Section 14.6) performance shall be computed and
maintained.
5.10.2.3 Data processing: The data system shall be used to search, locate, identify,
and quantify the compounds of interest in each GC analysis. Software
routines shall be employed to compute and record retention times and peak
areas. Displays of chromatograms and library comparisons are required to
verify results.
Halide-specific: Electron capture or electrolytic conductivity (Micoulometric,
Hall, 0.1., or equivalent), capable of detecting 100 pg of 2,4-D under the
analysis conditions given in Table 2.
5.9.1
5.9.2
5.9.3
5.9.4
5.10.3 Detectors.
5.10.3.1
725
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Method 1658
6. REAGENTS AND STANDARDS
6.1 Sample preservation: Sodium thiosulfate (ACS), granular.
6.2 pH adjustment.
6.2.1 Sodium hydroxide: Reagent grade.
6.2.1.1 Concentrated solution (ION): Dissolve 40 g NaOB in 100 mL reagent water.
6.2.1.2 Dilute solution (0.1M): Dissolve 4 g NaOH in 1 L of reagent water.
6.2.2 Sulfuric acid (1 + 1): Reagent grade, 6N in reagent water. Slowly add 50 mL 11 2 S0 4
(specific gravity 1.84) to 50 mL reagent water.
6.2.3 Potassium hydroxide: 37% (W/V). Dissolve 37 g KOH in 100 inL reagent water.
6.3 AcidifIed sodium sulfate: Add 0.5 mL 112504 and 30 mL ethyl ether to 100 g sodium sulfate.
Mix thoroughly. Allow the ether to evaporate completely. Transfer the mixture to a clean
container and store at 110°C (±5°C).
6.4 Solvents: Methylene chloride, hexane, ethyl ether, acetone,acetonitrile, isooctane, and methanol;
pesticide-quality; lot-certified to be free of interferences.
6.4.1 Ethyl ether must be shown to be free of peroxides before it is used, as indicated by EM
Laboratories Quant test strips (Scientific Products P1126-8, or equivalent). Procedures
recommended for removal of peroxides are provided with the test strips. After cleanup,
20mLofethylalcoholisaddedtoeachliterofether as apreservative.
6.5 GPC calibration solution: Solution containing 300 mg/mL corn oil, l5-mg/mL bis(2-ethylhexyl)
pbthalate, 1.4 mg mL pentachlorophenol, 0.1 mg/mL perylene, and 0.5 mg/mL sulfur.
6.6 Sample cleanup.
6.6.1 Plorisil: PR grade, 60/100 mesh, activated at 650 to 700°C, stored in the dark in glass
container with FFFE-lined screw-cap. Activate at 130°C for 16 hours minimum
immediately prior to use. Alternatively, 500-mg cartridges (J.T. Baker, or equivalent)
— used.
6.6.2 Solid-phas%extraction.
6.6.2.1 SPE cartridge calibration solution: 2,4,6-trichlorophenol, 0.1 &g/mL in
acetone.
6.6.2.2 SPE elution solvent: Methylene chloride: acetonitrile: bexane (50:3:47).
6.7 Dedvatization: Diazald reagent [ N -methyl -(N -nitroso -p -toluenesulfanamide)], fresh and high-
purity (Aldrich Chenucal Co.).
6.8 Reference matrices.
6.8.1 Reagent watec Water in which the compounds of interest and interfering compounds are
not detected by this method.
6.8.2 High-solids reference matrix: Playground sand or similar material in which the
compounds of Interest and interfering compounds are not detected by this method. May
be prepared by extraction with methylene chloride and/or baking at 450°C for 4 hours
mmn m•
726
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Method 1658
6.9 Standard solutions: Purchased as solutions or mixtures with certification to their purity,
concentration, and authenticity, or prepared from materials of known purity and composition. If
compound purity is 96% or greater, the weight may be used without correction to compute the
concentration of the standard. When not being used, standards are stored in the dark at
-20 to -10°C in screw-capped vials with PTFE-lined lids. A mark is placed on the vial at the
level of the solution so that solvent evaporation loss can be detected. The vials are brought to
room temperature prior to use. Any precipitate is redissolved and solyent is added if solvent loss
has occurred.
6.10 Preparation of stock solutions: Prepare in isooctane per the steps below. Observe the safety
precautions in Section 4.
6.10.1 Dissolve an appropriate amount of assayed reference material in solvent. For example,
weigh 10 mg 2,4-D in a 10-mL ground- glass stoppered volumetric flask and fill to the
mark with isooctane. After the 2,4-D is completely dissolved, transfer the solution to a
15-mL vial with PTFE-lined cap.
6.10.2 Stock standard solutions should be checked for signs of degradation prior to the
preparation of calibration or performance test standards.
6.10.3 Stock standard solutions shall be replaced after 6 months, or sooner if comparison with
quality control check standards indicates a change in concentration.
6.11 Secondary mixtures: Combine lock solutions (Section 6.10) into a secondary mixture at the
highest level required for required for calibration (Table 3). Derivatize the acids in this solution
using the procedure in Section 12. After derivatization, prepare the solutions for calibration and
calibration verification (Table 3), for initial and ongoing precision and recovery (Sections 8.2
and 14.6), and for spiking into the sample matrix (Section 8.4).
6.12 Surrogate spiking solution: Prepare 2,4-dichlorophenylacetic acid at a concentration of 2 ng/mL
in acetone.
6.13 Stability of solutions: All standard solutions (Sections 6.9 - 6.12) shall be analyzed within 48
hours of preparation and on a monthly basis thereafter for signs of degradation. Standards will
remain acceptable if the peak area remains within ±15% of the area obtained in the initial analysis
of the standard.
7. SETUP AND CAliBRATiON
7.1 ConfIgure the GC system as given in Section 5.10 and establish the operating conditions in
Table 2.
7.2 Attainment of method detection limit (MDL): Determine that the MDLs in Table 2 can be met
on each column/detector system.
7.3 Calibration: Inject the calibration solutions into each GC column/detector pair, beginning with
the lowest level mixture and proceeding to the highest. For each compound, compute and store,
as a function of the concentration injected, the retention time, and the peak area on each
column/detector system (primary and confirmatory).
7.3.1 Retention time: The polar nature of some analytes causes the retention time to decrease
as the quantity injected increases. To compensate this effect, the retention time for
compound identification is correlated with the analyte level.
727
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Method 1658
7.3.1.1 If the difference between the maximum and minimum retention times for any
compound is less than 5 seconds over the calibration range, the retention time
for that compound can be considered constant and an average retention-time
may be used for compound identification.
7.3.1.2 Retention time calibration curve (retention time vs. amount): If the retention
time for a compound in the lowest level standard is more than 5 seconds
greater than the retention time for the compound in the highest level standard,
a retention time calibration curve shall be used for identification of that
7.3.2 Calibration factor (ratio of area to amount injected).
7.3.2.1 Compute the coefficient of variation (relative standard deviation) of the
calibration factor over the calibration range for each compound on each
coiumW w system.
7.3.2.2 Linearity: If the calibration factor for any compound is constant (C, < 20%)
over the calibration range, an average calibration factor may be used for that
compound; ntherwise, the complete calibration curve (area vs. amount) for
that compound shall be used.
7.4 Combined QC standards: To preclude periodic analysis of all of the individual calibration groups
of compounds (Section 7.3.1), the (3C systems are calibrated with combined solutions as a final
step. NotallofthecompoundsinthesestandardswilbeseparatedbytheGCcOlnmnsUsed in
this method. Retention times and calibration factors are verified for the compounds that are
resolved, and calibration factors are obtained for the unresolved peaks. These combined QC
Standards are prepared at the level the mid-range calibration standard (Table 3).
7.4.1 Analyze the combined QC standards on their respective column/detector pairs.
7.4.1.1 For those compounds that exhibit a single, resolved GC peak,the retention
time shall be within ±5 seconds of the retention time of the peak in the
medium level calibration standard (Table 3), and the calibration factor using
the primary column shall be within ±20% of the calibration factor in the
iedium level standard (Table 3).
7.4.1.2 For the peaks containing two or more compounds, compute and store the
retention times at the peak maxima on both columns (primary and
confirmatory), and also compute and store the calibration factors on both
columns. These results will be used for calibration verification (Section 14.2
and 14.5) and for precision and recovery studies (Sections 8.2 and 14.6).
7.5 Flodsil callbratloà The cleanup procedure in Section 11 utilizes Florisil column chromatography.
Flotisil from different batches or sources may vary in adsorptive capacity. To standardize the
MK* of Flodsil that Is used, the use of the lauric acid value (Reference 4) is suggested. The
tth enoed procedure determines the adsorption of lauric acid (in milligrams per gram Florisil)
from beams solution. The amewt of Florisil to be used for each column is calculated by dividing
110 by this ratio and multiplying by 20 g.
728
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Method 1658
8. QuAun CONTROL
8.1 Each laboratory that uses this method Is required to operate a formal quality control program
(Reference 5). The minimum requirements of this program consist of an initial demonstration of
laboratory capability, an ongoing analysis of standards and blanks as tests of continued
performance, and analysis of spiked samples to assess accuracy. Laboratory performance is
compared to established performance criteria to determine if the results of analyses meet the
performance characteristics of the method. If the method is to be applied routinely to samples
containing high solids with very little moisture (e.g., soils, compost), the high-solids reference
matrix (Section 6.8.2) is substituted for the reagent water (Section 6.8.1) in all performance tests,
and the high-solids method (Section 10) is used for these tests.
8.1.1 The analyst shall make an initial demonstration of the ability to generate acceptable
accuracy and precision with this method. This ability is established as described in
Section 8.2.
8.1.2 The analyst is permitted to modify this method to improve separations or lower the costs
of measurements, provided all performance requirements are met. Each time a
modification is made to the method or a cleanup procedure is added, the analyst is
required to repeat the procedure in Section 8.2 to demonstrate method performance. If
detection limits will be affected by the modification, the analyst is required to repeat the
demonstration of detection Ihnits (Section 7.2).
8.1.3 The laboratory shall spike all samples with at least one surrogate compound to monitor
method performance. This test is described in Section 8.3. When results of these spikes
indicate a typical method performance for samples, the samples are diluted to bring
method performance Within acceptable limits (Section 17).
8.1.4 The laboratory shall, on an ongoing basis, demonstrate through calibration verification
and the analysis of the combined QC standard (Section 7.4) that the analysis system is
in control. These procedures are described in Sections 14.1, 14.5, and 14.6.
8.1.5 The laboratory shall maintain records to define the quality of data that is generated.
Development of accuracy statements is described in Section 8.4.
8.1.6 Analyses of blanks are required to demonstrate freedom from contamination. The
procedures and criteria for analysis of a blank are described in Section 8.5.
8.1.7 Other analytes may be determined by this method. The procedure for establishing a
preliminary quality control limit for a new analyte is given in Section 8.6.
8.2 Initial precision and recovery: To establish the ability to generate acceptable precision and
recovery, the analyst shall perform the following operations.
8.2.1 For analysis of samples containing low solids (aqueous samples), extract, concentrate,
and analyze one set of four 1-L aliquots of reagent water spiked with the combined QC
standard (Section 7.4) according to the procedure in Section 10. Alternatively, sets of
four replicates of the individual calibration groups (Section 7.3) may be used. For
samples containing high-solids, sets of four 30-g aliquots of the high-solids reference
matrix are used.
8.2.2 Using results of the set of four analyses, compute the average percent recovery (X) and
the coefficient of variation (Ce) of percent recovery (s) for each compound.
729
-------
Method 1658
8.2.3 For each compound, compare s and X with the corresponding limits for initial precision
and accuracy in Table 4. For coeluting compounds, use the coeluted compound with the
least restrictive specification (largest C. and widest range). If s and X for all compounds
meet the acceptance criteria, system performance is acceptable and analysis of blanks and
samples may begin. If, however, any individual s exceeds the precision limit or any
individual X falls outside the range for accuracy, system performance is unacceptable for
that compound. In this case, correct the problem and repeat the test.
8.3 The laboratory shall spike all samples with at least one surrogate compound to assess method
performance on the sample matrix.
8.3.1 Analyze each sample according to the method beginning in Section 10.
8.3.2 Compute the percent recovery (P) of the surrogate compound(s).
8.3.3 The recovery of the surrogate compound shall be within the limits of 40 to 120%. If the
recovery of any surrogate falls outside of these limits, method performance is
unacceptable for that sample, and the sample is complex. Water samples are diluted, and
smaller amounts of soils, sludges, and sediments are reanalyzed per Section 17.
8.4 Method accuracy: The laboratory shall spike (matrix spike) at least 10% of the samples from a
given site type (e.g., influent to treatment, treated effluent, produced water, river sediment). If
only one sample from a given site type is analyzed, that sample shall be spiked.
8.4.1 The concentration of the matrix spike shall be determined as follows.
8.4.1.1 If, as in compliance monitoring, the concentration of a specific analyte in the
s nple is being checked against a regulatory concentration limit, the matrix
spike shall be at that limit or at 1 to 5 times higher than the background
concentration determined in Section S 4.2, whichever concentration is larger.
8.4.1.2 If the concentration of an analyse in the sample is not being checked against
a limit specific to that analyte, the matrix spike shall be at the concentration
of the combined QC standard (Section 7.4) or at ito 5 times higher than the
background concentration, whichever concentration is larger.
8.4.1.3 if it is impractical to determine the background concentration before spiking
(e.g., maximum holding times will be exceeded), the matrix spike
concentration shall be the regulatory concentration limit, if any; otherwise,
the larger of either 5 times the expected background concentration or at the
concentration of the combined QC standard (Section 7.4).
8.4.2 Analyze one sample aliquot to determine the background concentration (B) of each
analyse. If necessary, prepare a standard solution appropriate to produce a level in the
sample one to five times the background concentration. Spike a second sample aliquot
with the standard solution and analyze it to determine the concentration after spiking (A)
of each analyte. Calculate the percent recovery (P) of each analyte:
730
-------
Method 1658
Equation 1
l0O(A-B )
T
where
T = True value of the spike
8.4.3 Compare the percent recovery for each analyte with the corresponding QC acceptance
criteria in Table 4. If any analyte fails the acceptance criteria for recovery, the sample
is complex and must be diluted and reanalyzed per Section 17.
8.4.4 As part of the QC program for the laboratory, method accuracy for samples shall be
assessed and records shall be maintained. After the analysis of five spiked samples of a
given matrix type (water, soil, sludge, sediment) in which the analytes pass the tests in
Section 8.4.3, compute the average percent recovery (P) and the standard deviation of
the percent recovery (s ) for each compound (or coeluting compound group). Express
the accuracy assessment as a percent recovery interval from P - 2s to P + 2 s for each
matrix. For example, if P = 90% and s = 10% for five analyses of compost, the
accuracy interval is expressed as 70 to 110%. Update the accuracy assessment for each
compound in each matrix on a regular basis (e.g., after each five to ten new accuracy
measurements).
8.5 Blanks: Reagent water and high-solids reference matrix blanks are analyzed to demonstrate
freedom from contamination.
8.5.1 Extract and concentrate a 1-L reagent water blank or a 30-g high-solids reference matrix
blank with each sample lot (samples started through the extraction process on the same
8-hour shift, to a maximum of 20 samples). Analyze the blank immediately after analysis
of the combined QC standard (Section 14.6) to demonstrate freedom from contamination.
8.5.2 If any of the compounds of interest (Fable 1) or any potentially interfering compound is
found in an aqueous blank at greater than 0.05 ig/L, or in a high-solids reference matrix
blank at greater than 1 gig/kg (assuming the same calibration factor as 2,4-D for
compounds not listed in Table 1), analysis of samples is halted until the source of
contamination is eliminated and a blank shows no evidence of contamination at this level.
8.6 Other analytes may be determined by this method. To establish a quality control limit for an
analyte, determine the precision and accuracy by analyzing four replicates of the analyte along
with the combined QC standard per the procedure in Section 8.2. If the analyte coelutes with an
analyte in the QC standard, prepare a new QC standard without the coeluting component(s).
Compute the average percent recovery (A) and the standard deviation of percent recovery (se) for
the analyte, and measure the recovery and standard deviation of recovery for the other analytes.
The data for the new analyte is assumed to be valid if the precision and recovery specifications
for the other analytes are met; otherwise, the analytical problem is corrected and the test is
repeated. Establish a preliminary quality control limit of A ± 2s for the new analyte and add
the limit to Table 4.
731
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Method 1658
8.7 The specifications contained in this method can be met if the apparatus used is calibrated properly,
then maintained in a calibrated state. The standards used for calibration (Section 7), calibration
verification (Section 14.5), and for initial (Section 8.2) and ongoing (Section 14.6) precision and
recovery should be identical, so that the most precise results will be obtained. The GC
instruments will provide the most reproducible results if dedIcated to the settings and conditions
required for the analyses of the analytes given in this method.
8.8 Depending on specific program requirements, field replicates and field spikes of the analytes of
interest into samples may be required to assess the precision and accuracy of the sampling and
sample transporting techniques.
9. SAMPLE COLLECTiON, PRESERVATiON, AND HANDLING
9.1 Collect samples in glass containers following conventional sampling practices, except that the
bottle shall not be prerinsed with sample before collection. Aqueous samples which flow freely
are collected in refrigerated bottles using automatic sampling equipment. Solid samples are
collected as grab samples using wide-mouth jars.
9.2 MaIntain samples at 0 to 4°C from the time of collection until extraction. If the samples will not
be extracted within 72 hours of collection, adjust the sample to a pH of less than 2 using sulfuric
acid solution. Record the volume of acid used. Caution: some samples require acidification in
a hood because of the potential for generating hydrogen sulfide.
9.3 If residual chlorine is present in aqueous samples, add 80 mg sodium thiosulfate per liter of water.
EPA Methods 330.4 and 330.5 may be used to measure residual chlorine (Reference 7).
9.4 Begin sample extraction within 7 days of collection, and analyze all extracts within 28 days of
10. SIMPLE EXTRACTION AND CONCENTRA T1ON
Samples containIng 1% solids or less are extracted directly using continuous liquid/liquid extraction
techniques (Section 10.2.1). Samples containing 1 to 30% solids are diluted to the 1 % level with reagent
water (Section 10.2.2) and extracted using continuous liquid-liquid extraction techniques. Samples
containing greater than 30% solids are extracted by tumbling with water in a rotary agitation apparatus.
The aqueous phase is then extracted using continuous liquid-liquid extraction techniques. Figure 1
outlines the extraction and concentration steps.
10.1 Determination of percent solids.
10.1.1 Weigh 5 to lOg of sample into a tared beaker. Record the weight to three significant
figures.
10.1.2 Dry overnight (12 hours minimum) at 110°C (±5°C), and cool in a desiccator.
10.1.3 Determine percent solids as follows:
EquatIon 2
% wiws = weight of dry sample ioo
weight of wet sample
732
-------
Method 1658
10.2 Preparation of samples for extraction.
10.2.1 Samples containing 1% solids or less.
10.2.1.1 Measure 1.00 L (±0.01 L) of sample into a clean 1.5- to 2.0-L beaker.
10.2.1.2 Spike 0.5 mL of the surrogate spiking solution (Section 6.12) into the sample
aliquot.
10.2.1.3 Proceed to preparation of the QC aliquots for low-solids samples
(Section 10.2.3).
10.2.2 Samples containing I to 30% solids.
10.2.2.1 Mix sample thoroughly.
10.2.2.2 Using the percent solids found in Section 10.1.3, determine the weight of
sample required to produce 1 L of solution containing 1% solids as follows:
Equation
3
1000
% solids
=
grains
10.2.2.3 Place the weight determined in Section 10.2.2.2 in a clean 1.5- to 2.0-L
beaker. Discard all sticks, rocks, leaves, and other foreign material prior to
weighing.
102.2.4 Bring the volume of the sample aliquot(s) to 400 to 500 mL with reagent
water.
10.2.2.5 Spike 0.5 mL of the surrogate spiking solution (Section 6.12) into each
sample aliquot.
10.2.2.6 Using a clean metal spatula, break any solid portions of the sample into small
pieces.
10.2.2.7 Place the %-inch horn on the ultrasonic probe approximately inch below
the surface of each sample aliquot and pulse at 50% for 3 minutes at full
power. If necessary, remove the probe from the solution and break any large
pieces using the metal spatula or a stirring rod and repeat the sonication.
Clean the probe with 5% aqueous sodium bicarbonate and then
methylenechioride: acetone (1:1) between samples to prevent damage to the
horn and preclude cross-contamination.
10.2.2.8 Bring the sample volume to 1.0 L (±0.1 L) with reagent water.
10.2.3 Preparation of QC aliquots for samples containing low solids (less than 30%).
10.2.3.1 For each sample or sample batch (to a maximum of 20) to be extracted at the
same time, place two 1.0 L (±0.01 L) aliquots of reagent water in clean
1.5- to 2.0-L beakers. Acidify to pH to less than 2 with HCI.
733
-------
Method 1658
10.2.3.2 Blank spike 0.5 mL of the surrogate spiking solution (Section 6.12) into one
reagent water aliquot.
10.2.3.3 Spike the combined QC standard (Section 7.4) into a reagent water aliquot.
10.2.3.4 If a matrix spike is required, prepare an aliquot at the concentrations specified
in Section 8.4.
10.2.4 Hydrolysis of acid esters and flocculation of particulates.
10.2.4.1 WhIle on astirringplate, raise the pH of the sample and QC aliquots to pH
12 to 13.
10.2.4.2 Stir and equilibrate all sample and QC solutions for 1 to 2 hours. Check the
p11 after approximately 0.5 hour and adjust if necessary.
10.2.4.3 Add sufficient NaC1 to saturate the solution. Approximately 350 g are
required. Stir to dissolve.
10.2.4.4 If the solution appears cloudy, add 2 g (±0.2 g) of CaCI 2 and allow to stand
for approximately 10 minutes to flocculate particulates.
10.2.4.5 Pre.extract the samples and QC aliquots to remove interferents per
Section 10.3.
10.2.5 Samples containing 30% solids or greater.’
10.2.5.1 Mix the sample thoroughly.
10.2.5.2 WeIgh 30 g (±0.3 g) of sample into a clean tumbler bottle. Discard all
sticks, rocks, leaves, and other foreign material prior to weighing.
10.2.5.3 Add l000m(±lOOmL)ofreagentwaterandadjustthepHto 12to l3using
NiOR.
10.2.5.4 QC aliquots: For each s*mple or sample batch (to a maximum of 20) to be
eatracted at the same time, place two 30 g (±0.3-g) aliquots of the high-
solids reference matrix in tumbler bottles. One aliquot will serve as the
blank.
10.2.5.5 Spike 0,5 mL of the surrogate spiking solution (Section 6.12) into each
aliquot.
10.2.5.6 To serve as the ongoing precision and recovery standard,spike 1.0 mL of the
combined QC standard (Section 7.4) into the remaining aliquot. Raise the pH
oftheQCaliquotsto l2to 13.
10.2.5.7 Tightly cap the tumbler bottles and tumble for 2 to 4 hours.
10.3 Pre es actlon to re ve laterferents: Place 100 to 150 mL methylene chloride in each continuous
estractor and 200 to 300 mL in each distilling flask.
10.3.1 uf the sample(s), blank , and standard aliquots into the extractors.
10.3.1.1 If a precipitate formed in the flocculation step (Section 10.2.4.4), or if the
sample contains other solids, pour the sample through filter paper into the
c 1ract o r.
734
-------
Methoo 1658
10.3.1.2 Rinse the containers with 50 to 100 mL methylene chloride and add to the
respective extractors. For samples that were filtered, pour the rinse over the
residual sample in the filter funnel and drain into the respective extractor.
10.3.2 Verify that the pH of the water in the extractors is 12 to 13.
10.3.3 Begin the extraction by heating the flask until the methylene chloride is boiling. When
properly adjusted, one to two drops of methylene chloride per second will fall from the
condenser tip into the water. Test and adjust the pH of the waters during the first 1 to
2 hours of extraction. Extract for 2 to 4 hours.
10.3.4 After extraction, remove the distilling flask, discard the methylene chloride, and add a
fresh charge of methylene chloride to the flask.
10.4 Extraction.
10.4.1 Adjust the pH of the water in the extractors to less than 2 with sulfuric acid.
10.4.2 Test and adjust the pH of the waters during the first 1 to 2 hours of the extraction.
Extract for 18 to 24 hours.
10.4.3 Remove the distilling flask, estimate and record the volume of extract (to the nearest
100 inL), and pour the contents through a prerinsed drying column containing 7 to 10 cm
of acidified anhydrous sodium sulfate. Rinse the distilling flask with 30 to 50 mL of
methylene chloride and pour through the drying column. Collect the solution in a
500-niL K-D evaporator flask equipped with a 1O-mL concentrator tube. Seal, label, and
concentrate per Sections 10.5 and 10.6.
10.5 Macro concentration.
10.5.1 Concentrate the extracts in separate 500-mL K-D flasks equipped with 10-mL
concentrator tubes. Add one or two clean, acid-rinsed boiling chips to the flask and
attach a three-ball macro Snyder column. Prewet the column by adding approximately
I niL of methylene chloride through the top. Place the K-D apparatus in a hot water bath
so that the entire lower rounded surface of the flask is bathed with steam. Adjust the
vertical position of the apparatus and the water temperature as required to complete the
concentration in 15 to 20 minutes. At the proper rate of distillation, the balls of the
column will actively chatter but the chambers will not flood.
10.5.2 When the liquid has reached an apparent volume of 1 mL, remove the K-I) apparatus
from the bath and allow the solvent to drain and cool for at least 10 minutes.
10.5.3 For extracts to be cleaned up using GPC, remove the Snyder column and rinse the flask
and its lower joint into the concentrator tube with 1 to 2 niL of methylene chloride. A
5-mL syringe is recommended for this operation. Adjust the final volume to 10 niL and
proceed to GPC cleanup in Section 11.
10.5.4 For extracts to be cleaned up using the SPE cartridge, adjust the final volume to 5.0 niL -
for those that have been cleaned up using GPC, and to 10 mL for those that have not.
Proceed to SPE cleanup in Section 11.
10.6 Hexane exchange: Extracts containing acids to be derivatized, extracts to be subjected to Florisil
cleanup, and extracts that have been cleaned up are exchanged into hexane.
735
-------
Method 1658
10.6.1 Remove the Snyder column, add approximately 50 mL of hexane and a clean boiling
chip, and reattach the Snyder column. Concentrate the extract as in Section 10.5, except
use hexane to prewet the column. The elapsed time of the concentration should be 5 to
10 minutes.
10.6.2 Remove the Snyder column and rinse the flask and its lower joint into the concentrator
tube with lto2mLofhexane.
10.6.2.1 For extracts containing acids to be esterifled, adjust the final volume to
10 mL for those that have not been cleaned up by GPC, and to 5 mL for
those that have been cleaned up by GPC (the difference accounts for the 50%
loss in the GPC cleanup). Proceed to Section 12 for esteriflcation.
10.6.2.2 For extracts to be cleaned up using Florisil, adjust the final volume to 5 to
10 mL and proceed to Florisil cleanup in Section 11.
10.6.2.3 For extracts to be analyzed by GC (Section 13), adjust the final volume to
10 mL for those that have not been cleaned up by GPC, and to 5 mL for
those that have been cleaned up by GPC.
11. CLEANUP
11.1 Cleanup procedures may not be necessary for relatively clean samples (treated effluents, ground
water, drinking water). If particular circumstances require the use of a cleanup procedure, the
analyst may use any or all of the procedures below or any other appropriate procedure. However,
the analyst shall first repeat the tests in Section 8.2 to demonstrate that the requirements of
Section 8.2 ca be met using the cleanup procedure(s) as an integral part of the method. Figure 1
outlines the cleanup steps.
11.1.1 Gel permeation chromatography (Section 11.2) removes many high molecular weight
iat feieuts that cause GC column performance to degrade. It is used for all soil and
sedime’* extracts and may be used for water extracts that are expected to contain high
molecular weight organic compounds (e.g., polymeric materials, humic acids).
11.1.2 The solid-phase extraction cartridge (Section 11.3) removes polar organic compounds
such as phenols.
11.1.3 The Florisil column (Section 11.4) allows for selected fractionation of the herbicides and
will also dimIn e polar interferences.
11.2 Gel nermeetlon chromatography (GPC).
11.2.1 Column packing .
• 11.2.1.1 Place lOto 75 g of SX-3 Bin-beads in a 400-to 500-mL beaker
11.2.1.2 Cover the beads with methylene chloride and allow to swell overnight
(12 hours minin am) .
11.2.1.3 Transfer the swelled beads to the column and pump solvent through the
column, from bottom to top, at 4.5 to 53 mLlmin prior to connecting the
column to the detector.
11.2.1.4 After purging the column with solvent for 1 to 2 hours, adjust the column
head pressure to 7 to 10 psig, and purge for 4 to 5 hours to remove air.
736
-------
Method 1658
Maintain a head pressure of 7 to 10 psig. Connect the column to the
detector.
11.2.2 Column calibration.
11.2.2.1 Load 5 mL of the calibration solution (Section 6.5) into the sample loop.
11.2.2.2 Inject the calibration solution and record the signal from the detector. The
elution pattern will be corn oil, bis (2-ethyihexyl) phthalate,
pentachiorophenol, perylene, and sulfur.
11.2.2.3 Set the “dump time” to allow >85% removal of the corn oil and >85%
collection of the phthalate.
11.2.2.4 Set the “collect time” to the peak minimum between perylene and sulfur.
11.2.2.5 Verify the calibration with the calibration solution after every 20 extracts.
Calibration is verified if the recovery of the pentachiorophenol is greater
than 85%. If calibration is not verified, the system shall be recalibrated using
the calibration solution, and the previous 20 samples shall be re-extracted and
cleaned up using the calibrated GPC system.
11.2.3 Extract cleanup: (3PC requires that the column not be overloaded. The column specified
in this method is designed to handle a maximum of 0.5 gram of high molecular weight
material in a 5 mL extract. If the extract is known or expected to contain more
than 0.5 g, the extract is split into fractions for GPC and the fractions are combined after
elution from the column. The solids content of the extract may be obtained
gravimetrically by evaporating the solvent from a 50-FL aliquot.
11.2.3.1 Filter the extract or load through the filter holder to remove particulates.
Load the 5.0-mL extract onto the column.
11.2.3.2 Elute the extract using the calibration data determined in Section 11.2.2.
Collect the eluate in a clean 400- to 500-mL beaker.
11.2.3.3 Rinse the sample loading tube thoroughly with methylene chloride between
extracts to prepare for the next sample.
11.2.3.4 If a particularly dirty extract is encountered, a 5.0-mL methylene chloride
blank shall be run through the system to check for carry-over.
11.2.3.5 Concentrate the extract and exchange to hexane per Section 10.6.
11.3 Solid-phase extraction (SPE).
11.3.1 Setup.
11.3.1.1 Attach the Vac-elute manifold to a water aspirator or vacuum pump with the
trap and gauge installed between the manifold and vacuum source.
11.3.12 Place the SPE cartridges in the manifold, turn on the vacuum source, and
adjust the vacuum to 5 to 10 psia.
11.3.2 Cartridge washing: Pre-elute each cartridge prior to use sequentially with 10-mL
portions each of hexane, methanol, and water using vacuum for 30 seconds after each
eluant. Follow this pre-elution with 1 mL methylene chloride and three 10-mL portions
of the elution solvent (Section 6.6.2.2) using vacuum for 5 minutes after each eluant.
Tap the cartridge lightly while under vacuum to dry between eluants. The three portions
737
-------
Method 1658
of elution solvent may be collected and used as a blank if desired. Finally, elute the
cartridge with 10 mL each of methanol and water, using the vacuum for 30 seconds after
each eluant.
11.3.3 Cartridge certification: E2c1i cartridge lot must be certified to ensure recovery of the
compounds of interest and removal of 2,4,6-trichiorophenol.
11.3.3.1 To make the test mixture, add the trichlorophenol solution (Section 6.6.2.1)
to the combined calibration standard (Section 7.4). Elute the mixture using
the procedure in Section 11.3.4.
11.3.3.2 Concentrate the eluant to 1.0 mL and inject 1.0 pL of the concentrated eluant
into the OC using the procedure in Section 13. The recovery of all analytes
(including the unresolved OC peaks) shall be within the ranges for recovery
specified in Table 4, and the peak for trichiorophenol shall not be detectable;
otherwise the SPE cartridge is not performing properly and the cartridge lot
shall be rejected.
11.3.4 Extract cIearr ip.
11.3.4.1 After cartridge washing (Section 11.3.2), release the vacuum and place the
rack coi ining the 50-mL volumetric flasks (Section 5.6.2.4) in the vacuum
m2nifold. Reestablish the vacuum at 5 to 10 psia.
11.3.4.2 Using a pipette or a 1-mL syringe, transfer 1.0 mL of extract to the SPE
cartridge. Apply vacuum for 5 minutes to dry the cartridge. Tap gently to
aid In drying.
11.3.4.3 Elide each cartridge into its volumetric flask sequentially with three 10-mL
portions of the elution solvent (Section 6.6.2.2), using vacuum for 5 minutes
after each portion. Collect the eluants in the 50-mL volumetric flasks.
11.3.4.4 Rele e the vacuum and remove the 50-mL volumetric flasks.
11.3.4.5 Using the nitrogen blow-down apparatus, concentrate the eluted extracts to
1.0 mL, and proceed to Section 13 for GC analysis.
11.4 Flodall colunm.
11.4.1 Place a weight of Florisil (nominally 20 g) predetermined by calibration (Section 7.5) in
a chrum*ogr hic column. T the column to settle the Florisil and add 1 to 2 cm of
aithydrous sodium sulfate to the top.
11.4.2 Add 60 mL of hexane to wet and rinse the sodium sulfate and Florisil. Just prior to
exposure of the sodium sulfate layer to the air, stop the elution of the hexane by closing
the stopcock on the chromatographic column. Discard the eluate.
11.4.3 Thnsfer the concentrated extract (Section 10.6.2) onto the column. Complete the
Uanef with t 1-mL hexane rinses.
738
-------
Method 1658
11.4.4 Place a clean 500-mL K-D flask and concentrator tube under the column. Drain the
column into the flask until the sodium sulfate layer is nearly exposed. Elute Fraction 1
with 200 mL of 6% ethyl ether in hexane (V/V) at a rate of approx 5 mL/min. Remove
the K-D flask. Elute Fraction 2 with 200 mL of 15% ethyl ether in hexane (V/V) into
a second K-D flask. Elute Fraction 3 with 200 mL of 50% ethyl ether in hexane (V/V).
11.4.5 Concentrate the fractions as in Section 10.6, except use hexane to prewet the column.
Readjust the final volume to 5 or 10 mL as in Section 10.6, depending on whether the
extract was subjected to GPC cleanup, and analyze by gas chromatography per the
procedure in Section 13.
12. ESTERIRCA liON
NOTE: Observe the safety precautions regarding diazomethane in Section 4.
12.1 Set up the diazomethane generation apparatus as given in the instructions in the Diazald kit.
12.2 Transfer I mL of the hexane solution containing the herbicides to a clean vial and add 0.5 mL of
methanol and 3 mL of ether.
12.3 Add 2 mL of diazomethane solution and let the sample stand for 10 minutes with occasional
swirling. The yellow color of diazomethane should persist throughout this period. If the yellow
color disappears, add 2 mL of diazomethane solution and allow to stand, with occasional swirling,
for another 10 minutes. Colored or complex samples will require at least 4 mL of diazomethane
to ensure complete reaction of the herbicides. Continue adding dizaomethane in 2-mL increments
until the yellow color persists for the entire 10-minute period or until 10 mL of diazomethane
solution has been added.
12.4.3 Rinse the inside waIl of the container with 0.2 to 0.5 mL of diethyl ether and add 10 to
20 mg of silicic acid to react excess diazomethane. Filter through Whatman #41 paper
into a clean sample vial. If the solution is colored or cloudy, evaporate to near dryness
using the nitrogen blowdown apparatus, bring to 1.0 mL with hexane, and proceed to
Section 11.3 for SPE cleanup. If the solution is clear and colorless, evaporate to near
dryness, bring to 1.0 mL with hexane and proceed to Section 13 for GC analysis.
13. GAS CHROMATOGRAPHY
NOTE: Table 2 swnmarizes the recommended operating conditions for the gas
chromoiograph. Included in this table are the retention times and estimated detection
limits that can be achieved under these conditions. Examples of the separations achieved
by the primary and confirmatory colwnns are shown in Figures 2 and 3.
13.1 Calibrate the system as described in Section 7.
739
-------
Method 7658
13.2 Set the injection volume on the auto-sampler to inject 1.0 L of all standards and extracts of
blanks and samples.
13.3 Set the data system or (3C control to start the temperature program upon sample injection, and
begin data collection after the solvent peak elutes. Set the data system to stop data collection alter
the last analyte is expected to elute and to return the column to the initial temperature.
14. SYSTEM AND LABORATORY PERFORMANCE
14.1 At the beginning of each 8-hour shift during which analyses are performed, GC system
performance and calibration are verified for all pollutants and surrogates on both column/detector
systems. For these tests, analysis of the combined QC standard (Section 7.4) shall be used to
verify all performance criteria. Adjustment and/or recalibration (per Section 7) shall be performed
until all peribrinance criteria are met. Only after all performance criteria are met may samples,
blanks, and precision and recovery standards be analyzed.
14.2 RetentIon times: The absolute retention times of the peak maxima shall be within ±10 seconds
of the retention times in the initial calibration (Section 7.4.1).
14.3 GC resolution: Resolution is acceptable if the valley height between two peaks (as measured from
the baseline) is less than 10% of the taller of the two peaks.
14.3.1 Primary column (DB-608): Dicamba and MCPA.
14.3.2 Confirmatory column (DB-1701): MCPP and MCPA.
14.5 CalIw*lon verification: Calibration is verified for the combined QC standard only.
14.5.t Inject the combined QC staiylard (Section 7.4)
14.5.2 Compute the percent recovery of each compound or coeluting compounds, based on the
calibration data (Section 7.4).
14.5.3 For each compound or coeluted compounds, compare this calibration verification
recovery with the corresponding limits for ongoing recovery in Table 4. For coeluting
compounds, use the coeluted compound with the least restrictive specification (the widest
range). If the recoveries for all compounds meet the acceptance criteria, system
performance is acceptable and analysis of blanks and samples may begin. If, however,
any recovery falls outside the calibration verification range, system performance is
unacceptable for that compound. In this case, correct the problem and repeat the test,
or recalibrate (Section 7).
14.6 Ongoing precision and recovery.
14.6.1 Analyze the extract of the precision and recovery standard extracted with each sample lot
(Secticea 10.2.3.3 and 10.2.5.7).
14.6.2 Compute the percent recovery of each analyte and coeluting compounds.
14.6.3 For each compound or coeluted compounds, compare the percent recovery with the limits
for ongoing recovery in Table 4. For coeluted compounds, use the coeluted compound
with the least restrictive specification (widest range). If all analytes pass, the extraction,
concentration, and cleanup processes are in control and analysis of blanks and samples
may proceed. If, however, any of the analytes fail, these processes are not in control.
740
-------
Method 1658
In this event, correct the problem, re-extract the sample batch, and repeat the on-going
precision and recovery test.
14.6.4 Add results which pass the specifications in Section 14.6.3 to initial and previous ongoing
data. Update QC charts to form a graphic representation of continued laboratory
performance. Develop a statement of laboratory data quality for each analyte by
calculating the average percent recovery (R) and the standard deviation of percent
recovery s,. Express the accuracy as a recovery interval from R - 2s to R + 25r. For
example, if R =95% and a, — 5%, the accuracy is 85 to 105%.
15. QUAUTA TIVE DETERMINA TION
15.1 Qualitative determination is accomplished by comparison of data from analysis of a sample or
blank with data from analysis of the shift standard (Section 14.2), and with data stored in the
retention-time and calibration libraries (Sections 7.3.2 and 7.3.3.2). Identification is confirmed
when retention time and amounts agree per the criteria below.
15.2 For each compound on each column/detector system, establish a retention-time window
±20 seconds on either side of the retention-time in the calibration data (Section 7.3.1). For
compounds that have a retention time curve (Section 7.3.1.2), establish this window as the
minimum -20 seconds and maximum +20 seconds.
15.2.1 Compounds not requiring a retention-time calibration curve: If a peak from the analysis
of a sample or blank is within a window (as defined in Section 15.2) on the primary
column/detector system, it is considered tentatively identified. A tentatively identified
compound is confirmed when (1) the retention time for the compound on the
confirmatory column/detector system is within the retention-time window on that system,
and (2) the computed amounts (Section 16) on each system (primary and confirmatory)
agree within a factor of 3.
15.2.2 Compounds requiring a retention-time calibration curve: If a peak from the analysis of
a sample or blank is within a window (as defined in Section 15.2) on the primary
column/detector system, it is considered tentatively identified. A tentatively identified
compound is confirmed when (1) the retention-times on both systems (primary and
confirmatory) are within ±30 seconds of the retention times for the computed amounts
(Section 16), as determined by the retention-time calibration curve (Section 7.3.1.2),
and (2) the computed amounts (Section 16) on each system (primary and confirmatory)
agree within a factor of 3.
16. QUAN77TA liVE DETERMINA 170N
16.1 Using the GC data system, compute the concentration of the analyte detected in the extract (in
micrograms per milliliter) using the calibration factor or calibration curve (Section 7.3.3.2).
16.2 Liquid samples: Compute the concentration in the sample using the following equation:
741
-------
Method 1658
Equation 4
c ,=1o :
where
= The concentration in the sample, in ig/L
10 The extract total, in niL
= The concentration in the extract, in j gImL
V , = The sample extracted, in L
16.3 Solid samples: Compute the concentration in the solid phase of the sample using the following
equation:
Equation 5
c=io (C )
1000(W,)(solidr)
where
C, Concentration in the sample, in &g/kg
10 Extract total, In niL
C = Concentration in the extract, In igImL
1000 = Convel7lon factor, g to kg
W, - Sample weight, in g
solids = Percent solids In Section 10.1.3 divided by 100
16.4 If the concentration of any analyte exceeds the calibration range of the system, the extract is
diluted by a factor of 10, and a 1- 1 sL aliquot of the diluted extract is analyzed.
16.5 Report result for all pollutants found in all standards, blanks, and samples to three significant
figures . Results for samples that have been diluted are reported at the least dilute level at which
the concentration is In the calibration range.
17. ANAL VaS OF COMPLEX SAMPLES
17.1 Some samples may contain high levels (>1000 ngIL) of the compounds of interest, interfering
compounds, and/or polymeric materials. Some samples may not concentrate to 10 mL
(SectIon 10.6); others may overload the GC column and/or detector.
17.2 The analyst shall attempt to clean up all samples using GPC (Section 11.2), the SPE cartridge
(Section 11.3), and flodsll (Section 11.4). If these techniques do not remove the interfering
compounds, the extract is diluted by a factor of 10 and reanalyzed (Section 16.4).
742
-------
Method 1658
17.3 Recovery of surrogates: in most samples, surrogate recoveries will be similar to those from
reagent water or from the high-solids reference matrix. If the surrogate recovery is outside the
range specified in Section 8.3, the sample shall be reextracted and reanalyzed. If the surrogate
recovery is still outside this range, the sample is diluted by a factor of 10 and reanalyzed
(Section 16.4).
17.4 Recovery of matrix spikes: In most samples, matrix spike recoveries will be similar to those from
reagent water or from the high-solids reference matrix. If the matrix spike recovery is outside
the range specified in Table 4, the sample shall be diluted by a factor of 10, respiked, and
reanalyzed. If the matrix spike recovery is still outside the range, the method does not work on
the sample being analyzed and the result may not be reported for regulatory compliance purposes.
18. METHOD PERFORMANCE
18.1 Development of this method is detailed in References 9 and 10.
743
-------
Method 1658
References
1. “Carcinogens: Working with Carcinogens.” Department of Health, Education, and Welfare;
Public Health Service; Center for Disease Control; National Institute for Occupational Health and
Safety: Publication 77-206, Aug 1977.
2. “OSHA Safety and Health Standards, General Industry” (29 CFR 1910). Occupational Safety and
Health Administration: Jan 1976.
3. “Safety in Academic Chemistry Laboratories,” American Chemical Society Committee on
Chemical Safety: 1979.
4. Mills, P. A., “Variation of Florisil Activity: Simple Method for Measuring Adsorbent Capacity
and Its Use in Standardizing Florisil Columns,” Journal of the Association of Official Analytical
Chemists, 51, 29: 1968.
5. “Handbook of Quality Control in Wastewater Laboratories,” U.S. Environmental Protection
Agency, Environmental Monitoring and SupportLaboratory, Cincinnati, OH,: EPA-600/4-79-019,
March 1979.
6. “Standard Practice for Sampling Water” (ASTM Annual Book of Standards), American Society
for Testing and Materials, Philadelphia, Pennsylvania: 76, 1980.
7. “Methods 330.4 and 330.5 for Total Residual Chlorine,” U.S. Environmental Protection Agency,
Environmental Monitoring and Support Laboratory, Cincinnati, OH: EPA-600/4-70-020, March
1979.
8. Jackson, Cary B. and Workman, Stephen M., “Analysis of Chiorophenoxy-Acid Herbicides in
Soil and Water,” presented at the 14th Annual EPA Conference on the Analysis of Pollutants in
the Environment, Norfolk, Virginia: May 1991.
9. “Consolidated GC Method for the Determination of ITDIRCRA Pesticides using Selective GC
Detectors,” S-CUBED, A Division of Maxwell Laboratories, Inc., La Jolla, CA: Ref. 32145-01,
Document RiO, September 1986.
10. “Method Development and Validation, EPA Method 1618, Cleanup Procedures,” Colorado State
University, Colorado Pesticide Center: November 1988 and January 1989.
744
-------
Method 1658
Table 1. Phenoxyacid Herbicides Determined by Large-Bore, Fused-Silica
Capillary Column Gas Chromatography with Halide-Specific Detector
EPA EGO Compound CAS Registry
481 2,4-D 94-75-7
480 Dinoseb 88-85-7
482 2,4,5-T 93-76-5
483 2,4,5-TP 93-72-1
Other phenoxyacid herbicides that can be analyzed by this method:
Compound CAS Registry
Dalapon 75-99-0
2,4-DB (Butoxon) 94-82-6
Dicamba 1918-00-9
Dich lorprop 120-36-5
MCPA 94-74-6
MCPP 7085-19-0
745
-------
Method 1658
Table 2. Gas chromatography of Phenoxy-Acid Herbicides
EPA Retention Time (m i i i ’ Method Detection Limit 2
EGD CompOwid D8408 DB-1707 (ng/L)
481 2,4-D 16.57 16.39 100
480 Dinoseb 20.75 23.55 50 (est)(ECD)
482 2,4,5-T 20.42 20.25 50
483 2,4,5-1 ? (Silvex) 18.65 18.66 40
Dalapon 3.52 3.63 100 est
2,4DB (Butoxon) 21.94 21.87 50
Dicamba 13.51 12.97 110
Dichlorprop 15.21 15.19 40
MCPA 14.42 14.30 90
MCPP 13.51 13.49 56
2,4-DCPA (surrogate) 12.88 12.51
Notes:
1. Cohinme: 30 at kmg by 053 mm ID, i.e., DB-608: 0.83 i; DB-1701: 1.0 i. Conditions suggested
to meet retention times shown: 175 to 270°C at 5°C/win., 175 to 2700 5°C/win. Carrier gas flow
rates approxim*Iy 7 mLImIn.
2. 40 Q7 Part 136, Appendix B (49 FR 43234). Detection limits for soils (in nglkg) are estimated to
be30to lOOtimesthis level.
746
-------
Method 1658
Table 3. Concentrations of Calibration Solutions
EPA Concentration (ng/mL)
EGD Compound Low Medium High
481 2,4-D 100 1,000 10,000
Dalapon 50 500 5,000
2,4-DB 200 2,000 20,000
2,4-DCPA (Surrogate) 10 1,000 10,000
Dicainba 20 200 2,000
Dichlorprop 100 1,000 10,000
480 Dinoseb 50 500 5,000
MCPA 5,000 50,000 500,000
MCPP 5,000 50,000 500,000
Picloram 50 500 5,000
482 2,4,5-T 20 200 2,000
483 2,4,5-TP (Silvex) 20 200 2,000
Eiectrolytic Conductivity Detector
481 2,4-1) 500 5,000 50,000
Dalapon 500 5,000 50,000
2,4-DB 1,000 10,000 100,000
2,4-DCPA (surrogate) 500 5,000 50,000
Dicamba 500 5,000 50,000
Dichlorprop 500 5,000 50,000
480 Dinoseb No Response
MCPA 500 5,000 50,000
MCPP 500 5,000 50,000
482 2,4,5-T 500 5,000 50,000
483 2,4,5-TP (Silvex) 250 2,500 25,000
747
-------
Method 1658
Table 4. Acceptance Criteria for Performance Tests for Phenoxy-Acid
Compounds
Acceptance criteria
Recovery
Spike and accuracy Calibration Ongoing
level (%) verification accuracy
EGO No. Compowzd (ng/L) X (pg/mU R (%)
481 2,4-D 200 16 41-107 78-121 23-13 1
480 Dinosth 500 18 24-154 64-136 19-159
482 2,4,5-T 100 17 30-132 70-130 5-158
483 2,4,5-TP (Silvex) 100 14 36-120 75-126 15-141
Dalapon 500 15 43-137 74-125 39-140
2,4-DB(j3utoxon) 100 22 22-118 42-157 0-142
Dicamba 200 18 37-145 59-139 10-172
Dichiorprop 100 14 49-133 71-128 28-154
MCPA 200 14 46-130 67-132 25-151
MCPP 400 14 65-149 71-129 42-170
Pidoram 500 13 46-140 73-126 42-144
748
-------
SpIke Surrogate
I _____ I
Figure 1. Extraction, Cleanup, Derivatization, and Analysis
Method 1658
Percent Solids 1
<30 Percent
r D II. To 1% SolIds
I 3O Percent
Spike Surrogate
Hydrolyze Esters
Hydrolyze Esters
Tumble With Water
Extract With CH 2 CI 2
Aqueous
Saturate With NaCI
I
PH<2I
Extract With CH 2 CI 2
Organic IPhase
Concentrate
FIOnSII Cleanup
GC/ECD
A52-O -82
749
-------
M.thrjd 1068
Silvex (18.86)
Uon Thw ss)
FIgure 2. Chromatogram of Herbicides DB-608 Column
D on 3.51)
Dicw*a4ACPP
He b
2,4,5T (20.43)
2,4,-D8 (21.94)
•1
0 5 10 15 20
25
30
M2 O2 .?9
750
-------
Method 1658
Silvex (18.7)
/
2, 4, 5-T (20.3)
/
2, 4-OB (21.9)
/
(23.6)
/
Chromatogram of Herbicides (DB-1 701 Column)
Dalapon (3.6)
/
Dicamba (13.0)
Herb Surr (12.5)
(9.9)
0.0 5.0 10.0 15.0 20.0 25.0
Retention Time (minutes)
30.0
Figure 3.
A52-002-80
751
-------
Method 1659
The Determination of Dazomet
in Municipal and Industrial
Waste water
-------
754
-------
Method 1659
The Determination of Dazomet in Municipal and Industrial
Waste water
1. SCOPE AND APPLICATION
1.1 This method covers the determination of dazomet (CAS 533-74-4) by base hydrolysis to methyl
isothiocyanate (MITC; CAS 556-61-6) and subsequent determination of MITC by wide bore,
fused-silica column gas chromatography (GC) with a nitrogen-phosphorus detector (NPD).
1.2 This method is designed to meet the monitoring requirements of the U.S. Environmental
Protection Agency under the Clean Water Act at 40 CFR Part 455. Any modification of this
method beyond those expressly permitted shall be considered a major modification subject to
application and approval of alternative test procedures under 40 CFR 136.4 and 136.5.
1.3 When this method is applied to analysis of unfamiliar samples, compound identity must be
supported by at least one additional qualitative technique. This method describes analytical
conditions for a second gas chromatographic column that can be used to confirm measurements
made with the primary column. Gas chromatography mass spectrometry (GCIMS) can be used
to confirm dazomet in extracts produced by this method when the level is sufficient.
1.4 The detection limit of this method is usually dependent on the level of interferences rather than
instrumental limitations. The limit in Table 1 typifies the minimum quantity that can be detected
with no interferences present.
1.5 This method is for use by or under the supervision of analysts experienced in the use of a gas
chromatograph and in the interpretation of gas chromatographic data. Each laboratory that uses
this method must demonstrate the ability to generate acceptable results using the procedure in
Section 8.2.
2. SUMMARY OF METHOD
2.1 A 50-mL sample is adjusted to pH 10 to 12 and allowed to stand for 3 hours to hydrolyze dazomet
to MITC. After hydrolysis, the sample is saturated with salt and extracted with 2.5 mL of ethyl
acetate. Gas chromatographic conditions are described that permit the separation and
measurement of MITC in the extract by wide-bore, fused-silica capillary column with
nitrogen-phosphorus detector (GCINPD).
2.2 Identification of MILTC (qualitative analysis) is performed by comparing the GC retention time of
the MITC on two dissimilar columns with the respective retention times of an authentic standard.
Compound identity is confirmed when the retention times agree within their respective windows.
2.3 Quantitative analysis is performed using an authentic standard of M1TC to produce a calibration
factor or calibration curve, and using the calibration data to determine the concentration of MITC -
in the extract. The concentration in the sample is calculated using the sample volume, the extract
volume, and a factor to convert MITC to dazomet.
2.4 Quality is assured through reproducible calibration and testing of the extraction and GC systems.
755
-------
Method 1659
3. CONTAMINATION AND INTERFERENCES
3.1 Solvents, reagents, glassware, and other sample-processing hardware may yield artifacts and/or
elevated baselines causing misinterpretation of chromatograms. All materials used in the analysis
shall be demonstrated to be free from interferences under the conditions of analysis by running
method blanks as described in Section 8.4.
3.2 Glassware and, where possible, reagents are cleaned by rinsing with and baking at 450°C for
1 hour minimum in a muffle furnace or kiln. Some thermally stable materials, such as PCBs, may
not be eliminated by this treatment and thorough rinsing with acetone and pesticide-quality hexane
may be required.
3.3 Specific selection of reagents and purification of solvents by distillation in all-glass systems may
be required.
3.4 Interferences coextracted from samples will vary considerably from source to source, depending
on the diversity of the site being sampled.
4. SAFETY
41 The toxicity or carcinogenicity of each compound or reagent used in this method has not been
precisely determined; however, each chemical compound should be treated as a potential health
h.z id. Exposure to these compounds should be reduced to the lowest possible level. The
laboratory is responsible for maintaining a current awareness file of OSHA regulations regarding
the safe haivfling of the chemicals specified in this method. A reference file of material handling
sheets should also be made available to all personnel involved in these analyses. Additional
inform ion on laboratory safety can be found in References 1 through 3.
4.2 Unknown samples may contain high concentrations of volatile toxic compounds. Sample
containers should be opened in a hood and handled with gloves that will prevent exposure.
5. APt’ARA TUS AND MA TERIALS
NOTE: Brand armies, s qqilkrs, and part nwnbers are for ilhsrtratlw purposes only. Th
endorsement I, inq. iied. Equivalent pesfonnance may be achieved using apparatus and
wi#erlth ether than those specified here, bat demonstration of equivalent pe!fonnance
meeting the tequimnents of tiILr method Lr the responsibility of the laboratosy.
5.1 SamplIng equipment for discrete or composite sampling.
5.1.1 Sample bottle: Amber glass, 1-L, with screw-cap. If amber bottles are not available,
uncles shaft be protected from light.
5.1.2 Bottle caps: Threaded to fit sample bottles. Caps shall be lined with FifE .
5 1.3 Qewilng.
5.1.3.1 Bottles are detergent water washed, then rinsed with solvent or baked at
450°C for 1 hour minimum before use.
5.1.3.2 Liners are detergent water washed, then rinsed with reagent water and
solvent, and baked at approximately 200°C for 1 hour minimum prior to use.
756
-------
Method 1659
5.1.4 Compositing equipment: Automatic or manual compositing system incorporating glass
containers cleaned per bottle cleaning procedure above. Sample containers are kept at
0 to 4°C during sampling. Glass or PTFE tubing only shall be used. If the sampler uses
a peristaltic pump, a minimum length of compressible silicone rubber tubing may be used
in the pump only. Before use, the tubing shall be thoroughly rinsed with methanol,
followed by repeated rinsings with reagent water to minimize sample contamination. An
integrating flow meter is used to collect proportional composite samples.
5.2 Extraction bottle: 4 oz. with PTFE-lined screw-cap, cleaned by solvent rinse or baking at 450°C
for 1 hour minimum.
5.3 pH meter, with combination glass electrode.
5.4 Sample vials: Amber glass, 1- to 5-mL with PTFE-lined screw- or crimp-cap, to fit GC
autosampler.
5.5 Balance: Analytical, capable of weighing 0.1 mg.
5.6 Miscellaneous glassware.
5.6.1 Pipettes, glass, volumetric, 1.00-, 5.00-, and 1O.0-mL.
5.6.2 Pipettes, glass, Pasteur.
5.6.3 Volumetric flasks, 10.0-, 25.0-, and 50.O-mL.
5.7 Gas chromatograph: Shall have splitless or on-column simultaneous automated injection into
separate capillary columns with a nitrogen-phosphorus detector at the end of each column,
temperature program with isothermal holds, data system capable of recording simultaneous signals
from the two detectors, and shall meet all of the performance specifications in Section 12.
5.7.1 GC columns: Bonded-phase fused-silica capillary.
5.7.1.1 Primary: 30 m long (± 3 m) by 0.5 mm (± 0.05 mm) ID, DB-608 (or
equivalent).
5.7.1.2 Confirmatory: DB-1701, or equivalent, with same dimensions as primary
column.
5.7.2 Data system: Shall collect and record GC data, store GC runs on magnetic disk or tape,
process GC data, compute peak areas, store calibration data including retention times and
calibration factors, identify GC peaks through retention times, compute concentrations,
and generate reports.
5.7.2.1 Data acquisition: GC data shall be collected continuously throughout the
analysis and stored on a mass storage device.
5.7.2.2 Calibration factors and calibration curves: The data system shall be used to
record and maintain lists of calibration factors, and multi-point calibration
curves (Section 7). Computations of relative standard deviation (coefficient
of variation) are used for testing calibration linearity. Statistics on initial
(Section 8.2) and ongoing (Section 12.5) performance shall be computed and
maintained.
5.7.2.3 Data processing: The data system shall be used to search, locate, identify,
and quantify the compounds of interest in each GC analysis. Software
routines shall be employed to compute and record retention times and peak
757
-------
Method 165.9
areas. Displays of chromatograms and library comparisons are required
to
verify results.
5.7.3 Nitrogen phosphorus detector: Thermionic bead or alkali flame detector, capable
of
detecting 600 pg of MITC under the analysis conditions given in Table 1.
6.
REAGENTS AND STANDARDS
NOTE: &w,d names, siqpliers, and part nwnbers are for illustrative pwposes only.
M, erdorsement Lc implied. Eqldwzlentpelfr7nance may be achieved using apparatus and
materials other than those specified here, but demonstration of equivalent performance
meeting the requirements of this method is the responsibility of the laboratory.
6.1 Se preservation: Sodium thiosulfate (ACS), granular.
6.2 pH adJustment.
6.2.1 Sodium hydroxide (iON): Dissolve 40 g NaOH in 100 mL reagent water.
6.2.2 Sulfuric acid (1 + 1): Reagent grade, 6N in reagent water. Slowly add 50 mL H 2 S0 4
(specific gravity 1.84) to 50 mL reagent water.
6.2.3 Potassium hydroxide: 31% (W/V). Dissolve 37 g KOH in 100 mL reagent water.
6.3 SoIv s: Methylene chloride, ethyl acetate, and acetone; pesticide-quality; lot-certified to be free
of haea1e ences.
6.4 Reagent water: Water in which the compounds of interest and interfering compounds are not
detected by this method.
6.5 Salt Sodium chloride, spread approximately 1 cm deep in a baking dish and baked at 450°C for
1 hour mininrnm
6.6 &aidard solutions: Purchased as solutions or mixtures with certification to their purity,
concentration, and authenticity, or prepared from materials of known purity and composition. If
cOiiçOwKl purity is 96% or greater, the weight may be used without correction to compute the
eeoc ntrM1on of the standard When not being used, standards are stored in the dark at -20 to
-10°C in screw-capped vials with Ffl E-Iined lids A mark is placed on the vial at the level of
the SolUtion so that solvent evaporation loss can be detected. The vials are brought to room
I .mpka.tw epr acrtouse.
8.7 PreparatIon of s solutions: Prepare in ethyl acetate per the steps below. Observe the safety
precw*lons In Section 4.
6.7.1 DIssolve an appropriate amount of assayed reference material in solvent For example,
weigh 10 mg MITC in a 10-niL ground-glass stoppered volumetric flask and fill to the
mark with ethyl acetate After the MITC is completely dissolved, transfer the solution
to a 15-mt vial with P’fl E-1ined cap
6.7.2 Stocksolutlonsshouldbechecked for signs of degradation prior to the preparation of
calibration or performance test standards.
6.7.3 Stock solutions shall be replaced after 6 months, or sooner if comparison with quality
control check standards indicates a change in concentration.
758
-------
Method 1659
6.8 Secondary mixtures: Using stock solutions (Section 6.7), prepare mixtures for calibration and
calibration verification (Sections 7.3 and 12.4), for initial and ongoing precision and recovery
(Sections 8.2 and 12.5), and for spiking into the sample matrix (Section 8.3).
6.8.1 Calibration solutions: Prepare MITC in ethyl acetate at concentrations of 0.2, 1.0, and
5.0 ig/mL. The midpoint solution (1.0 zgImL) is used for calibration verification
(Section 12.4).
6.8.2 Precision and recovery standard: Prepare M1TC in acetone at a concentration of
25 pg/rnL.
6.8.3 Matrix spike solution: Prepare dazomet in acetone at a concentration of 25 gImL.
6.9 Stability of solutions: All standard solutions (Sections 6.7 and 6.8) shall be analyzed within
48 hours of preparation and on a monthly basis thereafter for signs of degradation. Standards will
remain acceptable if the peak area remains within ± 15% of the area obtained in the initial analysis
of the standard.
7. SETUP AND CAUBRA T1ON
71 Configure the GC system as given in Section 5.7 and establish the operating conditions in Table 1.
7.2 Attainment of minimum level: Determine that each column/detector system meets the minimum
level for MITC (Fable 1).
7.3 Calibration.
7.3.1 Inject 3 L of each calibration solution (Section 6.8.1) into each GC column/detector
pair, beginning with the lowest level mixture and proceeding to the highest. For each
compound, compute and store, as a function of the concentration injected, the retention
time and peak area on each column/detector system (primary and confirmatory).
7.3.2 Calibration factor (ratio of area to amount injected).
7.3.2.1 Compute the coefficient of variation (relative standard deviation) of the
calibration factor over the calibration range for MITC on each
column/detector system.
7.3.2.2 Linearity: If the calibration factor is constant (C < 20%) over the
calibration range, an average calibration factor may be used; otherwise, the
complete calibration curve (area vs. amount) shall be used.
8. QuAliTY CONTROL
8.1 Each laboratory that uses tins method is required to operate a formal quality control program. 4
The minimum requirements of this program consist of an initial demonstration of laboratory
capability, an ongoing analysis of standards and blanks as tests of continued performance, and
analysis of spiked samples to assess accuracy. Laboratory performance is compared to established
performance criteria to determine if the results of analyses meet the performance characteristics
of the method.
8.1.1 The analyst shall make an initial demonstration of the ability to generate acceptable
accuracy and precision with this method. This ability is established as described in
Section 8.2.
759
-------
Mettwd 1659
8.1.2 The analyst is permitted to modify this method to improve separations or lower the costs
of measurements, provided all performance requirements are met. Each time a
modification is made to the method or a cleanup procedure is added, the analyst is
required to repeat the procedure in Section 8.2 to demonstrate method performance. If
detection the detection limit for dazomet will be affected by the modification, the analyst
is required to repeat demonstration of the detection limit (Section 7.2).
8.1.3 The laboratory shall, on an ongoing basis, demonstrate through calibration verification
and the analysis of the precision and recovery standard (Section 6.8.2) that the analysis
system is in control. These procedures are described in Sections 12.1, 12.4, and 12.5.
8.1.4 The laboratory shall m2it in records to define the quality of data that is generated.
Development of accuracy statements is described in Section 8.3.
8.1.5 Analyses of blanks are required to demonstrate freedom from contamination The
procedures and criteria for analysis of a blank are described in Section 8.4.
8.2 In i I precision and recovery: To establish the ability to generate acceptable precision and
acaara y, the analyst shall perform the following operations:
8.2.1 Extract, concentrate, and analyze one set of four 50-mL aliquots of reagent water spiked
wIth 0.1 mL of the precision and recovery standard (Section 6.8.2) according to the
procedure in Section 10.
8.2.2 Using results of the set of four analyses, compute the average percent recovery (X) and
the coefficient of variation (Ce) of percent recovery(s) for M1TC.
8.2.3 Compare a and X with the corresponding limit for initial precision and recovery in
Table 1. Ifs and X meet the ___criteria, system performance is acceptable and
analysis of blanks and s mpIes may begin. If, however, s exceeds the precision limit or
X falls outside the range for accuracy, system performance is unacceptable. In this case,
correct the problem and repeat the test.
8.3 M hod accuracy: The laboratory shall spike (matrix spike) at least 10% of the samples from a
given ike type (e.g., influeit to treatm , treated effluent, produced water). If only one sample
h m a given sitetype is analyzed, a separate aliquot of that sample shall be spiked.
8.3.1 The concentration of the matrix spike shall be determined as follows:
8.3.1.1 If, as in compliance monitoring, the concentration of dazomet in the sample
is being checked against a regulatory concentration limit, the matrix spike
shall be at that limit or at 1 to 5 times higher than the background
concentration determined in Section 8.3.2, whichever concentration is larger.
8.3.1.2 If the concentration is net being checked against a regulatory limit, the matrix
sp lkesh allbeat5 opg(Lorat lto5dmeshigherthanthebackground
concentration, whichever concentration is larger.
8.3.1.3 If it is impractical to determine the background concentration before spiking
(e.g., maxim.m holding times will be exceeded), the matrix spike
concentration shall be the regulatory concentration limit, if any; otherwise,
the larger of either 5 times the expected background concentration, or
50 gsg/L (the concentration produced by 0.1 mL of the matrix spike solution
spiked into a 50-mL sample).
‘ •b ’.-, P.)I b ;
760
-------
Method 1659
8.3.2 Analyze one sample aliquot to determine the background concentration (B) of dazomet.
If necessary, prepare a standard solution appropriate to produce a level in the sample 1 to
5 times the background concentration. Spike a second sample aliquot with the standard
solution and analyze it to determine the concentration after spiking (A) with dazomet.
Calculate the percent recovery (P):
Equation 1
= 100 ( A-B )
T
where
T= True value of the spike
8.3.3 Compare the percent recovery for dazomet with the corresponding QC acceptance criteria
in Table 1. If dazomet fails the acceptance criteria for recovery, the sample is complex
and must be diluted and reanalyzed per Section 15.
8.3.4 As part of the QC program for the laboratory, method accuracy for samples shall be
assessed and records shall be maintained. After the analysis of five spiked samples of
a given matrix type (treated effluent, influent to treatment, produced water) in which the
recovery test (Section 8.3.3) is passed, compute the average percent recovery (P) and the
standard deviation of the percent recovery (se). Express the accuracy assessment as a
percent recovery interval from P - 2 s to P + 2s for each matrix. For example, if
P 90% and s, = 10% for five analyses of wastewater, the accuracy interval is
expressed as 70 to 110%. Update the accuracy assessment in each matrix on a regular
basis (e.g., after each five to ten new accuracy measurements).
8.4 Blanks: Reagent water blanks are analyzed to demonstrate freedom from contamination.
8.4.1 Extract and concentrate a 50-mL reagent water blank with each sample batch (samples
started through the extraction process on the same 8 hour shift, to a maximum of 20
samples). Analyze the blank immediately after analysis of the precision and recovery
standard (Section 12.5) to demonstrate freedom from contamination.
8.4.2 If MITC or any potentially interfering compound is found in an aqueous blank at greater
than 2 1 zgfL (assuming the same calibration factor as MITC for interfering compounds),
analysis of samples is halted until the source of contamination is eliminated and a blank
shows no evidence of contamination at this level.
8.5 The specifications contained in this method can be met if the apparatus used is calibrated properly,
then maintained in a calibrated state. The standards used for calibration (Section 7), calibration
verification (Section 12.4), and for initial (Section 8.2) and ongoing (Section 12.5) precision and
recovery should be identical, so that the most precise results will be obtained. The GC instrument
will provide the most reproducible results if dedicated to the settings and conditions required for
the analyses of the analytes given in this method.
8.6 Depending on specific program requirements, field replicates and field spikes may be required to
assess the precision and accuracy of the sampling and sample transporting techniques.
761
-------
Method 1659
9. SAMPLE CouEc ThON, PRESERVATiON, AND HANDLING
9.1 Collect samples in glass containers following conventional sampling practices, 5 except that the
bottle shall not be prerinsed with sample before collection. Aqueous samples which flow freely
are collected in refrigerated bottles using automatic sampling equipment.
9.2 Maintain samples at 0 to 4°C from the time of collection until extraction. If the samples will not
be extracted within 72 hours of collection, adjust the sample to a pH greater than 9.0 using
sodium hydroxide solution. Record the volume used. If residual chlorine is present in aqueous
samples, add 80 mg sodium thiosulfate per liter of water. EPA Methods 330.4 and 330.5 may
be used to measure residual chlorine.’
9.3 Begin sample extraction within seven days of collection, and analyze all extracts within 40 days
of extraction.
10. SAMPLE HYDROi Y S AND EXTRACTiON
10.1 HydrolysIs and preparation of QC aliquots.
10.1.1 Pour 50 niL of sample into a dean 4-ox. bottle. If a matrix spike is to be prepared, pour
50 niL into a second clean bottle
10.1.2 For each s n le or sample batch (to a maximum of 20) to be extracted at the same time,
place two 50-niL aliquóts of reagent water in clean 4-oz. bottles. One reagent water
aliqix* serves as the blank.
10.1.3 Spike 0.1 ml. of the precision and recovery standard (Section 6.8.2) into the remaining
teag t water ot.
10.1.4 Spike 0.1 niL of the matrix spike solution (Section 6.8.3) into the sample aliquot used
for the matrix spike.
10.1.5 Test the pH of the sample and QC aliquots with a pH meter and adjust to 10 to 12 with
potassium hydroxide solution. Cap and shake the bottles vigorously to mix. Allow to
stand.
10.1.6 TestandadjustthepHaftero.5to 1 hour. Allowtostandforanadditional2to3hours.
10.1.7 Extract the sample and QC aliquots per Section 10.2.
102 Extraction.
10.2.1 Add 20gof clean Naa (Section 6.5) and 2.5 niL of ethyl acetate to each sample and QC
aliquot and cap tightly.
10.2.2 Shake vigorously for 2 to 5 mi!ujtes . Allow the bottle to stand for 10 minutes for the
phases to separate.
10.2.3 UsIng a Paste t pipette, transfer the organic phase to a GC autosampler vial. Measure
svoluns.
11. GAs CM OMATOGRAPHY
Table 1 in.. . a1 the recomnionded operating conditions for the gas chromatograph. Included in this
table is the rete J lion for M1TC achieved under these conditions. An example of the sei aration
achieved by the primary column is sbown in Figure 1.
762
-------
Method 1659
1 1.1 Calibrate the system as described in Section 7.
1 1.2 Set the injection volume on the autosampler to inject 3.0 jiL of all standards and extracts of blanks
and samples.
11.3 Set the data system or GC control to start the temperature program upon sample injection, and
begin data collection after the solvent peak elutes. Set the data system to stop data collection after
the last analyte is expected to elute and to return the column to the initial temperature.
12. SYSTEM AND LABORA TORY PERFORMANCE
12.1 At the beginning of each 8-hour shift during which analyses are performed, GC system
performance and calibration are verified on both column/detector systems. For these tests,
analysis of the calibration verification standard (Section 6.8.1) shall be used to verify all
performance criteria. Adjustment and/or recalibration (per Section 7) shall be performed until all
performance criteria are met. Only after all performance criteria are met may samples, blanks,
and precision and recovery standards be analyzed.
12.2 Retention times: The absolute retention time of the peak maxima shall be within ±10 seconds
of the retention times in the initial calibration (Section 7.3.1).
12.3 GC resolution: Resolution is acceptable if the peak width at half-height is less than 10 seconds.
12.4 Calibration verification.
12.4.1 Inject the calibration verification standard (Section 6.8.1).
12.4.2 Compute the concentration of MITC based on the calibration factor or calibration curve
(Section 7.3).
12.4.3 Compare this concentration with the limits for calibration verification in Table 1. If the
recovery meets the acceptance criteria, system performance is acceptable and analysis of
blanks and samples may begin. If, however, the recovery falls outside the calibration
verification range, system performance is unacceptable. In this case, correct the problem
and repeat the test, or recalibrate (Section 7).
12.5 Ongoing precision and recovery.
12.5.1 Analyze the extract of the precision and recovery standard extracted with each sample
batch (Section 10.1.3).
12.5.2 Compute the percent recovery of M1TC.
12.5.3 Compare the percent recovery with the limits for ongoing recovery in Table 1. If the
recovery meets the acceptance criteria, the extraction and concentration processes are in
control and analysis of blanks and samples may proceed. If, however, the recovery falls
outside the acceptable range, these processes are not in control. In this event, correct the
problem, re-extract the sample batch, and repeat the ongoing precision and recovery test.
12.5.4 Add results which pass the specifications in Section 12.5.3 to initial and previous ongoing
data. Update QC charts to form a graphic representation of continued laboratory
performance. Develop a statement of laboratory data quality for each analyte by
calculating the average percent recovery (R) and the standard deviation of percent
recovery s 1 . Express the accuracy as a recovery interval from R - 2s to R + 2sf. For
example, if R = 95% and S = 5%, the accuracy is 85 to 105%.
763
-------
Method 1659
13. QUALITA 71VE DETERMINATION
13.1 Qualitative determination is accomplished by comparison of data from analysis of a sample or
blank with data from analysis of the shift standard (Section 12.1), and with data stored in the
retention time and calibration libraries (Section 7.3.1). Identification is confirmed when retention-
time and amounts agree per the criteria below.
13.2 On each column/detector system, establish a retention-time window ±20 seconds on either side
of the retention-time in the calibration data (Section 7.3.1).
13.3 IftheMTrCpeakfromtheanalysisofasampleorblank iswithinawindow (as defined in
Section 13.2) on the primary column/detector system, it is considered tentatively identified.
A tentatively identified compound is confirmed when (1) the retention time for the compound on
the confirmatory column/detector system is within the retention-time window on that system, and
(2) the computed amounts (Section 14) on each system (primary and confirmatory) agree within
a factor of 3.
14. QuANtflATIvE DETERMINA liON
14.1 Using the GC data system, compute the concentration of the analyte detected in the extract (in
milligrams per milliliter) using the calibration factor or calibration curve (Section 7.3.2).
14.2 Compute the concentration in the sample using the following equation:
EqratI i 2
( 2 22XV)(C)
v
ç =
The conce vzratlon in the sample, in g g/L
2.22
Conwrt M1TC (MW 73.12) to dazomet (MW
162.27)
V
The e.uracz total volume, in mL
C =
The concentration in the &tract, In pglmL
V ’
Thew 1wneofswiq1etaracted,1nL
14.3 If the concentration of MITC exceeds the calibration range of the system, the extract is diluted
by a orof 10, and a 3-pL aliquot of the diluted extract is analyzed.
14.4 R *t ilealts for dazomet found in all standards, blanks, and samples to three significant figures.
Rendis for samples that have been diluted are reported at the least dilute level at which the
concentration is In the calibration range
15. *iIISLY S COMPLEX SAMPLES
15.1 *oiiic P9 4 .1 may co’ high levels (>1000 ng/L) of dazomet or of interfering compounds,
end/or polymeric materials Some samples may form emulsions when extracted (Section 102),
Others 111*7 overload the GC column and/or ddector In these instances, the extract is diluted by
a factor of 10 aMreanaly ed (Section 14.3).
764
-------
Method 1659
15.2 Recovery of matrix spikes: In most samples, matrix spike recoveries will be similar to those from
reagent water. If the matrix spike recovery Is outside the range specified in Table 1, the sample
is diluted by a factor of 10, respiked, and reanalyzed. If the matrix spike recovery is still outside
the range, the method may not work on the sample being analyzed and the result may not be
reported for regulatory compliance purposes.
16. METHOD PERFORMANCE
16.1 This method is based on industry Method 131.
16.2 Development of this method is detailed in Reference 8.
765
-------
* t?IoU 1b55
References
1. ‘Carcinogens: Working with Varciimgens.’ Department of Health, Education, and Welfare;
Public Health Service; Center for Disease Control; National Institute for Occupational Health and
Safety: Publication 77-206, Aug 1977.
2. OSHA Safety and Health Standards, General Industry’ (29 CFR 1910). Occupational Safety and
Health Administration: Jan 1976.
3. ‘Safety in Academic Chemistry Laboratories;’ American Chemical Society Committee on
Chemical Safety: 1979.
4. ‘Handbook of Quality Control in Wastewater Laboratories,’ U.S. Environmental Protection
Agency, Environmental Monitoring and Support Laboratory, Cincinnati, OH,: EPA-600/4-79-O 19,
M th 1979.
5. ‘ Stapdard Practice for Sampling Water’ (ASTM Annual Book of Standards), American Society
for Testing and Materials, Philadelphia, Pennsylvania: 76, 1980.
6. ‘Methods 330.4 and 330.5 for Total Residual Chlorine,’ U.S. Environmental Protection Agency,
Environmental Monitoring and Support Laboratory, Cincinnati, OH: EPA-600/4-70-020, March
1979.
7. ‘Determination of Metham (Vapam) in Wastewater’ (Method 131), Methods for Nonconvenrional
Penldde Qiemicalt Analysis of Induitriol and Mwzicipal Waste ner. U.S. Environmental
Protection Agency Effluent Guidelines Division (WH-552), Washington, DC:
EPA 440/1-83/079-C, J2nnary 31, 1983.
8. ‘N rMwe for SAS 1019,’ Pacific Analytical, Inc.: September 1989. Available from the U.S.
Environmental Protection Agency Sample Control Center, 300 N. Lee St., Mexandria, VA 22314
(103-557-5040).
766
-------
Method 1659
Tab’e 1. GC Data and Method Acceptance Criteria for Dazomet*
Acceptance Criterion Specification Note
Minimum Level 10 gIL 1
Method Detection Limit 3 gfL 2
Calibration Verification (Section 12.4) 0.8 to 1.3 gImL 3
Initial Precision and Recovery (Section 8.2)
Precision [ standard deviation; s] 23 j g/L
Recovery [ mean; X} 18 to 75 ig/L
Ongoing Precision and Recovery (Section 12.5) 15 to 78 g/L
Matrix Spike Recovery (Section 8.3.3) 16 to 123 %
MITC Retention-time 5
DB-608 2.17 minutes
DB-1701 3.80 minutes
*(3,5 im yl2Ht ydro.. 1 ,3,5-thiadiazine-2-thione) detected as Methyl isothiocyanate (MITC)
Notes:
1. This is a minimum level at which the analytical system shall give recognizable signals and
acceptable calibration points.
2. Estimated; 40 CFR Part 136, Appendix B.
3. Test concentration 1.0 igImL.
4. Test concentration 50 j gfL.
5. Columns: 30 mm long by 0.53 mm ID; DB-608: 0.83 DB-1701: 1.0 . Conditions suggested
to meet retention-times shown: 50°C for 1.0 minute, 50 to 200° at 10°C/mm. Carrier gas flow
rates approximately 7 mL/min.
767
-------
— 1 .
(0.6)
/
... w 17W flV. I 7 1.
/
(1.0)
/
(2.8) (5.4)
‘ IL iT I I I I I I I I I I I I I
0.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0
41
Figure 1. Chromatogram of Methyl isothiocyanate
-------
Method 1660
The Determination of Pyre thrins
and Pyre throids in Municipal
and Industrial Waste water
-------
770
-------
Method 1660
The Determination of Pyrethrins and Pyrethroids in Municipal and
Industrial Waste water
1. SCOPE AND APPLICA TION
1.1 This method covers the determination of pyrethrins and pyrethroids in wastewater by extraction
and high performance liquid chromatography (HPLC) with an ultra-violet detector (UV). The
compounds in Table 1 may be determined by this method.
1.2 This method is designed to meet the monitoring requirements of the U.S. Environmental
Protection Agency under the Clean Water Act at 40 CFR Part 455. Any modification of this
method beyond those expressly permitted shall be considered a major modification subject to
application and approval of alternative test procedures under 40 CFR 136.4 and 136.5.
1.3 When this method is applied to analysis of unfamiliar samples, compound identity must be
supported by at least one additional qualitative technique. This method lists a second UV
wavelength that can be used to confirm measurements made with the primary wavelength.
1.4 This method is specific to the determination of two pyrethrins and seven pyrethroids, but should
be applicable to other pyrethroids as well. The quality control requirements in this method give
the steps necessary to determine this applicability.
1.5 The detection limit of this method is usually dependent on the level of interferences rather than
instrumental limitations. The limits in Table 2 typify the minimum quantity that can be detected
with no interferences present.
1.6 This method is for use by or under the supervision of analysts experienced in the use of a high
performance liquid chromatograph and interpretation of liquid chromatographic data. Each
laboratory that uses this method must demonstrate the ability to generate acceptable results using
the procedure in Section 8.2.
2. SUMMARY OF METHOD
2.1 A 750-mL sample is saturated with salt and extracted by stirring with acetonitrile in a 1-L
volumetric flask. A small portion of the acetonitrile rises into the neck of the flask. 1 The extract
is evaporated to a volume of 7.5 mL.
2.2 A 40-iL aliquot of the extract is injected into the HPLC. Chromatographic conditions are
described that permit the separation and measurement of the pyrethrins and pyrethroids by reverse-
phase C18 column HPLC with a multiple-wavelength UV detector.
2.3 Identification of compound is performed by comparing the retention time of the compound with
that of an authentic standard. Compound identity is confirmed when the retention times agree,
and when the response at a second wavelength agrees with the response at the primary
wavelength.
2.4 Quantitative analysis is performed using an authentic standard of each compound to produce a
calibration factor or calibration curve, and using the calibration data to determine the
771
-------
Method 1660
concentration of that compound in the extract. The concentration in the sample is calculated using
the sample and extract volumes.
25 Quality is assured through reproducible calibration and testing of the extraction and HPLC
systems.
3. CoNrAFjI jA liON AND INTERFERENCES
3.1 Solvents, reagents, glassware, and other sample processing hardware may yield artifacts and/or
elevated baselines causing misinterpretation of chromatograms. All materials used in the analysis
shall be demonstrated to be free from interferences under the conditions of analysis by running
method bl nkc as described in Section 8,4.
3.2 Glassware and, where possible, reagents are cleaned by rinsing with solvent and baking at 450°C
lbr I hour mininuim in a muffle furnace or kiln. Some thermally stable materials may not be
elim’n ad by this treatment and thorough rinsing with acetone and pesticide-quality acetonitrile
may be required.
3.3 Specific selection of reagents and purification of solvents by distillation in all-glass systems may
be required.
3.4 interfrrences coextracted from samples will vary considerably from source to source, depending
on the diversity of the site being sampled.
4. S iv
4.1 The toxicity or carcinogenicity of each compound or reagent used in this method has not been
precisely determined; however, each chemical compound should be treated as a potential health
h igd. Exposure to these compounds should be reduced to the lowest possible level. The
laboratory is responsible for m ii ining a current awareness file of OSHA regulations regarding
the safe h 1Iing of the chemicals specified in this method. A reference file of material handling
sheets should also be made available to all personnel involved in these analyses. Additional
lnlbnn*ion on laboratory safety can be found in References 2 through 4.
4.2 Unknown samples may contain high concentrations of volatile toxic compounds. Sample
coi*ainers should be opened in a hood and handled with gloves that will prevent exposure
5 ArnRiuus MA TEFJALS
NO1E Irand aWnes, szçpVer c, and pan numbers are for ilhsstradvepwposes only. No
is Implied. EquMilent peiformwzce may be achieved using apparatus and
iufdx v.*Iser than those q ecffted here, but demonstration Qf equivalent performance
meeting reqidrenengs’ cf this method Is the responsibility qf the laboratory
5.1 S pling equipment for discrete or composite sampling.
5.1.1 Sample bottle Amber gl s , 1-L, with screw-cap If amber bottles are not available,
swiçles shall be protected from light
5.1.2 Bottle caps: Threaded to fit sample bottles. Caps shall be lined with PTFE.
772
-------
Method 1660
5.1.3 Cleaning.
5.1.3.1 Bottles are detergent water washed, then rinsed with solvent or baked at
450°C for 1 hour minimum before use.
5.1.3.2 Liners are detergent-water washed, then reagent water and solvent rinsed, and
baked at approximately 200°C for 1 hour minimum prior to use.
5.1.4 Compositing equipment: Automatic or manual compositing system incorporating glass
containers cleaned per bottle cleaning procedure above. Sample containers are kept
at 0 to 4°C during sampling. Glass or PTFE tubing only shall be used. If the sampler
uses a peristaltic pump, a minimum length of compressible silicone rubber tubing may
be used in the pump only. Before use, the tubing shall be thoroughly rinsed with
methanol, followed by repeated rinsings with reagent water to minimize sample
contamination. An integrating flow meter is used to collect proportional composite
samples.
5.2 Equipment for glassware cleaning.
5.2.1 Laboratory sink with overhead fume hood.
5.2.2 Kiln: Capable of reaching 450°C within 2 hours and holding 450°C within ±10°C, with
temperature controller and safety switch (Cress Manufacturing Co. Sante Fe Springs,
CA, B3 1H or X3 iTS, or equivalent).
5.3 Equipment for sample extraction.
5.3.1 Laboratory fume hood.
5.3.2 Stirring plate: Therinolyne Ciinarec 2 (Model 546725), or equivalent.
5.3.3 Stirring bar: PTFE coated, approximately 1 by 4 cm.
5.3.4 Extraction flask: l000-mL volumetric flask cleaned by rinsing with solvent or baking
at 450°C for 1 hour minimum.
5.3.5 pH meter, with combination glass electrode.
5.4 Equipment for sample concentration.
5.4.1 Nitrogen evaporation device: Equipped with heated bath that can be maintained at 35 to
40°C (N-Evap, Organomation Associates, Inc., or equivalent).
5.4.2 Concentrator tube: 10- to 15-mL, graduated (Kontes K-570050-1025, or equivalent) with
calibration verified.
5.5 Sample vials: Amber glass, 10- to 15-mL with PTFE-lined screw- or crimp-cap, to fit HPLC
autosampler.
5.6 Balance: Analytical, capable of weighing 0.1 mg.
5.7 Miscellaneous glassware.
5.7.1 Pipettes, glass, volumetric, 1.00-, 5.00-, and 10.0-mL.
5.7.2 Pipettes, glass, Pasteur, 150 mm long by 5 mm I I) (Fisher Scientific 13-678-6A, or
equivalent).
5.7.3 Volumetric flasks, 10.0-, 25.0-, and 50.0-mL
773
-------
Vathod 1660
5.8 High performance liquid chroinatograph (HPLC): Analytical system complete with pumps,
sample injector, column oven, and multiple-wavelength ultra-violet (UV) detector.
5.8.1 Pumping system: Capable of isocratic operation and producing a linear gradient
from 70% water/30% acetonitrile to 100% acetonitrile in 25 minutes (Waters 600E, or
equi ).
5.8.2 Sample injector: Capable of automated injection of up to 30 samples (Waters 700, or
5.8.3 Column oven: Capable of operation at room ambient to 50°C (Waters TCM, or
equivalent).
5.8.4 Colunni : No 150 inin 10mg by 4.6 mm ID 300 Angstrom C18 columns (Vydac 201
TP5415, or equivalent) connected in series and preceded by a 30 mm long by 4.6 mm
ID 300 Angstrom C18 guard column (Vydac 201 GCC54T, or equivalent), operated at
the coalitions shown in Table 2.
5.8.5 Datector: UV operated at 235 and 245 nm (Waters 490E, or equivalent).
5.9 Data system.
5.9.1 Data acquisition: The data system shall collect and record LC peak areas and retention
*In Ofl n4gfletic uiedlL
5.9.2 Calibration: The data system shall be used to calculate and n int in lists of calibration
ors (response divided by concentration) and multi-point calibration curves.
Computations of relative standard deviation (coefficient of variation) are used to test
calibration linearity.
5.9.3 Data processing: The data system shall be used to search, locate, identify, and quantify
the compounds of interest in each analysis. Displays of chromatograms are required to
5.9.4 Statistics on initi 1 (Section 8.2) and ongoing (Section 12.5) performance shall be
— and mInd hied .
6. AG 1TS STAAVARDS
6.1 Sample preservation: Sodium thiosulfate (ACS), granular.
6,2 Hadjà .
6.2.1 SodIum hydroxide (ION): Dissolve 40 g NaOH in 100 mL reagent water.
6.2.2 Sulfuric acid (1 + 1). Reagent grade, 6N in reagent water Slowly add 50 mL H 2 S0 4
(specific gravity 1.84) to 50 mL reagent water
6.3 Solve. s: Ace u. 1i’rile and acetone; pesticide-quality; lot-certified to be free of interferences.
6.4 Reagent wU ; HPLC grade water in which the compounds of interest and interfering compounds
detected by this method.
6.5 SaJt Sodium duI*xlde, spread proxhn*lyl cm deep in a baking dish and baked at 450°C for
I beur mm .Iv im cooled aid stored in a precleaned glass bottle with Pim-lined cap.
6.6 Standard solutions: Purchased as solutions or mixtures with certification to their purity,
concentration, aiKI authenticity, or prepared from materials of kjxwn purity and composition. If
774
-------
Method 1660
compound purity is 96% or greater, the weight may be used without correction to compute the
concentration of the standard.
NO TE: The pyretlzrins are normally available in a mixed standard consisting of the six
naturally occurring compounds (pyrethrin I and 11, cinerin I and If, and jasmolin land!!).
The concentrations in this standard will be on the order of 10% each ofpyrethrin land II.
The concentration in the stock solution prepared from this mixed standard is to be
corre cted for the exact concentration.
When not being used, standards are stored in the dark at -20 to -10°C in screw-capped
vials with PTFE-lined lids. A mark is placed on the vial at the level of the solution so that
solvent evaporation loss can be detected. The vials are brought to room temperature prior
to use.
6.7 Preparation of stock solutions: Prepare in acetonitrile per the steps below. Observe the safety
precautions in Section 4.
6.7.1 Dissolve an appropriate amount of assayed reference material in solvent. For example,
weigh 10-mg allethrin in a 1O-mL ground-glass stoppered volumetric flask and fill to the
mark with acetonitrile. After the allethrin is completely dissolved, transfer the solution
to a 15-mL vial with PTFE-lined cap.
6.7.2 Stock solutions should be checked for signs of degradation prior to the preparation of
calibration or performance test standards.
6.7.3 Stock solutions shall be replaced after 6 months, or sooner if comparison with quality
control check standards indicates a change in concentration.
6.8 Secondary mixtures: Using stock solutions (Section 6.7), prepare mixtures for calibration and
calibration verification (Sections 7.3 and 12.4), for initial and ongoing precision and recovery
(Sections 8.2 and 12.5), and for spiking into the sample matrix (Section 8.3).
6.8.1 Calibration solutions: Prepare two solutions in acetomtrile at the concentrations given
in Table 3. The midpoint solution is used for calibration verification (Section 12.4)
6.8.2 Precision and recovery standard and matrix spike solution: Prepare two solutions in
acetone at the concentration of the midpoint standard (Table 3).
6.9 Stability of solutions: All standard solutions (Sections 6.7 through 6.8) shall be analyzed within
48 hours of preparation and on a monthly basis thereafter for signs of degradation. Standards will
remain acceptable if the peak area remains within ± 15% of the area obtained in the initial analysis
of the standard.
7. SETUP AND CALIBRATION
7.1 Configure the HPLC system as given in Sections 5.8 through 5.9 and establish the operating
conditions in Table 2.
7.2 Attainment of minimum level: Determine that the minimum levels in Table 2 are met at each
wavelength.
7.3 Calibration.
775
-------
Vai’hod 1660
7.3.1 Inject 40 ,&L of each calibration solution (Table 3) into the HPLC system, beginning with
the lowest level mixture and proceeding to the highest. For each compound, compute
and store, as aflinction of the concentration injected, the retention time and the peak area
at each wavelàngth (primary and confirmatory).
7.3.2 Calibration factor (ratio of area to amount h jected).
7.3.2.1 Comput. the coefficient of variation (relative standard deviation) of the
calibration factor over the calibration range for each at each wavelength.
7.3.2.2 Linearity If the calibration factor is constant (C < 20%) over the
Calibration range, an average calibration factor may be used; otherwise, the
- complete calibration curve (area vs. amount) shall be used.
8. OawTv CoNmoL
8.1 Eadi laboratory that uses this method is required to operate a formal quality control program.
The mlnlmi .m requiremeeta of this program consist of an initial demonstration of laboratory
jability, an ongoing analysis of standards and blanks as tests of continued performance, and
analysis of spiked samples to assess accuracy. Laboratory performance is compared to established
performance c k ia to detennine If the results of analyses meet the performance characteristics
of the method .
LII The analyst shall m’fr an initial demonstration of the ability to generate acceptable
accuracy and precision with this method. This ability is established as described in
Section 8.2.
8.1.2 The analyst is permitted to modify this method to improve separations or lower the costs
oi messwe’i s , provided all performance requirements are met. Each time a
is e to the method or a de procedure is added, the analyst is
reipdred torepest the procedure In Section 8.2 to demonstrate method performance. If
detection limits will be affected by the modification, the analyst is required to repeat
dasnenstratlon of the detection limit (Section 72)
813 L1 laboratory shall, on an ongoing basis, demonstrate through calibration verification
and the analysis of the precision and recovery standard (Section 6.8.2) that the analysis
system is in control. These procedures are described in Sections 12.1, 12.4, and 12.5.
8.1.4 The laboratory shall miint m records to define the quality of data that is generated
Devalopmm* of accuracy statements is described in Section 8.3.
8.1.5 Analyses of blanks are required to demonstrate freedom from contamination. The
procedures and cdt6ia for analysis of a blank are described in Section 8.4.
8.2 InitiaL precision and recovery: To establish the ability to generate acceptable precision and
e analyst shall perform the following operations:
8.2.1 Extract, concentrate, and analyze two sets of four 750-mL aliquots of reagent water
with 1.0 mL of each solution of the precision and recovery standard
(SIetlon 6.82) accordIng to the procedure in Section 10
8.2.2 Using results of each set of four analyses, compute the average recovery (X) and the
standard deviation of recovery (a), in milligrams per liter, for the each compound.
776
-------
Method 1660
8.2.3 Compare s and X with the corresponding limit for initial precision and recovery in
Table 4. If s and X meet the acceptance criteria, system performance is acceptable and
analysis of blanks and samples may begin. If, however, s exceeds the precision limit or
X falls outside the range for accuracy, system performance is unacceptable. In this case,
correct the problem and repeat the test.
8.3 Method accuracy: The laboratory shall spike (matrix spike) at least 10% of the samples from a
given site type (e.g., influent to treatment, treated effluent, produced water). If only one sample
from a given site type is analyzed, a separate aliquot of that sample shall be spiked.
8.3.1 The concentration of the matrix spike shall be determined as follows.
8.3.1.1 If, as in compliance monitoring, the concentration of allethrin in the sample
is being checked against a regulatory concentration limit, the matrix spike
shall be at that limit or at 1 to 5 times higher than the background
concentration determined in Section 8.3.2, whichever concentration is larger.
8.3.1.2 If the concentration is not being checked against a regulatory limit, the matrix
spike shall be at the level of the precision and recovery standard
(Section 6.8.2) or at 1 to 5 times higher than the background concentration,
whichever concentration is larger.
8.3.1.3 If it is impractical to determine the background concentration before spiking
(e.g., maximum holding times will be exceeded), the matrix spike
concentration shall be the regulatory concentration limit, if any; otherwise,
at the level of the precision and recovery standard (Section 6.8.2) or at I to
5 times the expected background concentration, whichever is larger.
8.3.2 Analyze one sample aliquot to determine the background concentration (B) of the
pyrethrins and pyrethroids. If necessary, prepare a standard solution appropriate to
produce a level in the sample 1 to 5 times the background concentration. Spike a second
sample aliquot with the standard solution and analyze it to determine the concentration
after spiking (A) of each analyte. Calculate the percent recovery (P):
Equation 1
= 100 ( A-B )
T
where
T = True value of the spike
8.3.3 Compare the percent recovery of each compound with the corresponding QC acceptance
criteria in Table 4. If any analyte fails the acceptance criteria for recovery, the sample
is complex and must be diluted and reanalyzed per Section 15.
8.3.4 As part of the QC program for the laboratory, method accuracy for samples shall be
assessed and records shall be maintained. After the analysis of five spiked samples of a
given matrix type (water, sludge) in which the recovery test (Section 8.3.3) is passed,
compute the average percent recovery (P) and the standard deviation of the percent
777
-------
Method 1660
recovery (s). Express the accuracy assessment as a percent recovery interval from P- 2s
toP + 2s,foreach matrix. For example, ifP = 90% ands = 10% for five analyses
of wastewater, the accuracy interval is expressed as 70 to 110%. Update the accuracy
assessment in each matrix on a regular basis (e.g., after each five to ten new accuracy
measurements).
8.4 Blanks: Reagent water blanks are analyzed to demonstrate freedom from contamination.
8.4.1 Extract and concentrate a 750-mL reagent water blank with each sample batch (samples
started through the extraction process on the same 8 hour shift, to a maximum of 20
samples). Analyze the blank immediately after analysis of the precision and recovery
standard (Section 12.5) to demonstrate freedom from contamination.
8.4.2 If any compound or any potentially interfering compound is found in an aqueous blank
at greater than 20 pg/L (assuming the same calibration factor as allethrin for interfering
compounds), analysis of samples is halted until the source of contamination is eliminated
and a blank shows no evidence of contamination at this level.
8.5 Other pyrethroids may be determined by this method. To establish a quality control limit for
another analyte, determine the precision and accuracy by analyzing four replicates of the analyte
along with the precision and recovery standard per the procedure in Section 8.2. If the analyte
coelutes with an analyte in the QC standard, prepare a new QC standard without the coeluting
um ponent(s). Compute the average percent recovery (A) and the standard deviation of percent
recovery (s) for the analyte, and measure the recovery and standard deviation of recovery for the
other analytes. The data for the new analyte is assumed to be valid if the precision and recovery
specifications for the other analytes are met; otherwise, the analytical problem is corrected and
the test is repeated. Establish a preliminary quality control limit of A ± 2s for the new analyte
andaddthelimittoTable4.
8.6 The specifications contained in this method can be met if the apparatus used is calibrated properly,
then m ainedIn a calibrated state. The standards used for calibration (Section 7), calibration
!,ef’lfjcation (Section 12.4), aM for initial (Section 8.2) and ongoing (Section 12.5) precision and
recovery should be identical, so that the most precise results will be obtained. The HPLC
instrument will provide the most reproducible results if dedicated to the settings and conditions
required for the analyses of the analytes given in this method.
8.7 Depending on specific program requirements, field replicates and field spikes may be required to
assess the precision and accuracy of the sampling and sample transporting techniques.
9. S4MPLE COLLECTION, PRESERVATION, AND HANDLING
9.1 Collect samples in glass containers following conventional sampling practices, 6 except that the
bottle shall a be prerinsed with sample before collection. Aqueous samples which flow freely
are colleoted In refrigerated bottles uwig automatic sampling equipment.
9.2 Mabiain ‘ es atOm 4°C from the time of collection until extraction. If the samples will not
be extracted within 72 hours of collection, adjust the sample to a pH of 5.0 to 7.0 using sodium
hydioxideor bydrodtloric acid solution. Record the volume used. If residual chlorine is present
in aqueous samples, add 80 mg sodium thiosulfate per liter of water. EPA Methods 330.4
and 330.5 may be used to measure residual chlorine. 7
778
-------
Method 1660
9.3 Begin sample extraction within 7 days of collection, and analyze all extracts within 40 days of
extraction.
10. SAMPLE EXTRACTiON
10.1 Preparation of sample and QC aliquots.
10.1.1 Mix sample thoroughly.
10.1.2 Pour 750 mL of sample into a clean 1000-mL volumetric flask. If a matrix spike is to
be prepared, pour two 750-mL aliquots into clean flasks.
10.1.3 For each sample or sample batch (to a maximum of 20) to be extracted at the same time,
place three 750-mL aliquots of reagent water (Section 6.4) in clean 1000-mL volumetric
flasks. One reagent water aliquot serves as the blank.
10.1.4 Spike 1.0 mL of each precision and recovery standard (Section 6.8.2) into the remaining
reagent water aliquots.
10.1.5 Spike the samples designated as the matrix spike at the level directed in Section 8.3.
10.1.6 Extract the sample and QC aliquots per Section 10.2.
10.2 Extraction.
10.2.1 Place each sample or QC aliquot on a stirring plate and add a clean PTFE-coated stirring
bar.
10.2.2 Add 230 g of clean NaCI (Section 6.5) to each sample and QC aliquot and stir 5 to 10
minutes to dissolve.
10.2.3 Add 160 mL of acetonitrile to each sample and QC aliquot and begin stirring. Increase
the rate of stirring until the vortex is drawn approximately one-half the depth of the
water. Stir for approximately 5 minutes.
10.2.4 Allow the solutions to stand for approximately 5 minutes for the phases to separate. If
an acetonitrile layer does not appear, add acetonitrile in 5-mL increments, stirring and
settling between increments, until a 2- to 5-mL layer appears. If the acetonitrile layer
is more than 5 mL, add reagent water, stir, and settle until the acetonitrile volume is
reduced to 2 to 5 niL.
10.2.5 Using a Pasteur pipette, transfer the organic phase to a clean K-I) concentrator tube
(Section 5.4.2).
10.2.6 Add 5 mL of acetonitrile to the extraction flasks, stir, and allow to settle. Transfer the
organic phase to the respective concentrator tubes. Repeat the extraction a third time.
If all of the extract will not fit into the concentrator tube, evaporate some of the
acetonitrile (Section 10.3), then add the remaining extract.
10.3 Concentration of extracts.
10.3.1 Place the concentrator tubes in the evaporation device (Section 5.4.1). Adjust the height
of the blow-down tubes to 1 to 3 cm above the surface of the liquid and gently evaporate
the acetonitrile until a volume of approximately 5 mL is reached.
10.3.2 Adjust the final extract volume to 7.5 mL and transfer to an HPLC autosampler vial.
77.9
-------
Method 1660
11. HIGH PERFORMANCE LIQWD CHROMATOGRAPHY
Table 2 summarizes the recommended operating conditions for the HPLC system. Included in this table
are the retention times for the pyrethrins and pyrethroids achieved under these conditions. An example
of the separation achieved by the column system is shown in Figure 1. Pyrethrin I and II are the major
peaks in the naturally occurring pyrethrin standard. Pyrethrin II elutes prior to pyrethrin I. Jasmolin II
and I will normally coelute with pyrethrin II and I, respectively. Most HPLC columns will resolve
cinerin II and I, which are small peaks that elute after the respective pyrethrins. Some HPLC columns
may resolve all six of the naturally occurring pyrethrins.
11.1 Calibrate the system as described in Section 7.
11.2 Set the injection volume on the autosampler to inject 40 uL of all standards and extracts of blanks
and samples.
11.3 Set the data system or HPLC control to start the gradient upon sample injection, and begin data
collection after 10 minutes . Set the data system or HPLC control to stop data collection after the
last analyte is expected to elute and to return the gradient to the initial setting.
12. SYSTEM AND LABORA TORY PERFORMANCE
12.1 At the beginning of each 8 hour shift during which analyses are performed, HPLC system
performance and calibration are verified at both wavelengths. For these tests, analysis of the
calilifation verification standard (Section 6.8.1) shall be used to verify all performance criteria.
Adjustment and/or recalibration (per Section 7) Shall be performed until all performance criteria
are met. Only after all perlbrmance criteria are met may samples, blanks, and precision and
recovery standards be analyzed.
12.2 Retention f es .
12.2.1 The absolute retention time of sumithrin shall be no earlier than 23 minutes.
12.2.2 The absolute retention time of the peak maxima shall be within ±15 seconds of the
average retention times in the initial calibration (Section 7.3.1).
12.3 GC resolution. Resolution is acceptable if the height of the valley between tetramethrm and
allaihrin is less than 20% of the taller of the two peaks when chromatograms of the two calibration
verification solutions (Section 6.8.1) are superimposed.
12.4 Calibration verification.
12.4.1 Inject the two calibration verification standards (Section 6.8.1).
12.4.2 Compute the concentration of the pyrethrins and pyrethroids based on the calibration
factor or calibration curve (Section 7.3).
124.3 Conipare this concentration with the limits for calibration verification in Table 4. If
calibration is verified, system performance is acceptable and analysis of blanks and
samples may begin. If, however, the recovery falls outside the calibration verification
range, system performance is unacceptable. In this case, correct the problem and repeat
the test, or recalibrate (Section 7)
12.5 Ongoing precision and recovery.
12.5 .1 Analyze the extract of the two precision and recovery standards extracted with each
sample batch (Section 10.1.3).
780
-------
Method 1660
12.5.2 Compute the recovery of the compounds of interest in milligrams per liter.
12.5.3 Compare the recovery with the limits for ongoing recovery in Table 4. If the recovery
meets the acceptance criteria, the extraction and concentration processes are in control
and analysis of blanks and samples may proceed. If, however, the recovery falls outside
the acceptable range, these processes are not in control. In this event, correct the
problem, re-extract the sample batch, and repeat the ongoing precision and recovery test.
12.5.4 Add results which pass the specifications in 12.5.3 to initial and previous ongoing data.
Update QC charts to form a graphic representation of continued laboratory performance.
Develop a statement of laboratory data quality for each analyte by calculating the average
percent recovery (R) and the standard deviation of percent recovery s 1 . Express the
accuracy as a recovery interval from R - 2Sr to R + 2sf. For example, if R = 95% and
= 5%, the accuracy is 85 to 105%.
13. QUALITATIVE DETERMINA liON
13.1 Qualititative determination is accomplished by comparison of data rom analysis of a sample or
blank with data from analysis of the shift standard (Section 12.1), and with data stored in the
retention-time and calibration libraries (Section 7.3.1). Identification is confirmed when retention
time and amounts agree per the criteria below.
13.2 Establish a retention-time window of ±20 seconds on either side of the mean retention -time in
the calibration data (Section 7.3.1).
13.3 If a peak from the analysis of a sample or blank is within a window (as defined in Section 13.2)
at the primary wavelength (235 rim), it is considered tentatively identified. A tentatively identified
compound is confirmed is confirmed when (1) the retention time of the peak maximum at the
confirmatory wavelength (245 mn) is within ±2 seconds of the retention-time of the peak
maximum at the primary wavelength, and (2) the computed amounts (Section 14) on each system
(primary and confirmatory) agree within a factor of 2.
14. QUANTITATiVE DETERMINA TION
14.1 Using the HPLC data system, compute the concentration of the analyte detected in the extract
(in micrograms per milliliter) using the calibration factor or calibration curve (Section 7.3.2).
14.2 Compute the concentration in the sample using the following equation:
Equation 2
= (V )(C )
vs
where
C =
The
concentration in the sample,
in p.gIL
V =
The
extract total volume, in mL
(nominally,
7.5)
C =
The
concentration in the extract,
in gIL
V 3 =
The
voiwne of sample extracted,
in L (nominally, 0.75)
781
-------
Method 1660
14.3 If the concentration of any analyte exceeds the calibration range of the system, the extract is
diluted by a factor of 10, and a 4O- iL aliquot of the diluted extract is analyzed.
14.4 Report results for pyrethrins and pyrethroids found in all standards, blanks, and samples to three
significant figures. Results for samples that have been diluted are reported at the least dilute level
at which the concentration is in the calibration range.
15. ANALYSES OF COMPLEX SAMPLES
15.1 Some sam$es may contain high levels (>1000 ng/L) of the pyrethrins and pyrethroids or of
interfering compounds and/or polymeric materials. Some samples may form emulsions when
extracted (Section 10.2); others may overload the HPLC column and/or detector. In these
Instances, the sample is diluted by a factor of 10 and re-extracted (Section 10), or the extract is
diluted by a factor of 10 and reanalyzed (Section 14.3).
15.2 Recovery of matrix spikes: In most samples, matrix spike recoveries will be similar to those from
reagent water . If the matrix spike recovery is outside the range specified in Table 4, the sample
Is diluted by a factor of 10, resplked, and reanalyzed. If the matrix spike recovery is still outside
thó range, the method does not work on the sample being analyzed and the result may not be
reported tbr regulatory cOmpliance purposes.
16. ME1$ PEm’0RM4NCE
16.1 Deveiopmest of this method is detailed in Reference 8.
782
-------
Method 1660
References
1. Leggett, Daniel F., Jenkins, T. F., and Miyares, P. H., Analytical Chemistry, pp 1355-1356:
July 1990.
2. “Carcinogens: Working with Carcinogens.” Department of Health, Education, and Welfare;
Public Health Service; Center for Disease Control; National Institute for Occupational Health and
Safety: Publication 77-206, Aug 1977.
3. OSHA Safety and Health Standards, General Industry’ (29 CFR 1910). Occupational Safety and
Health Administration: Jan 1976.
4. “Safety in Academic Chemistry Laboratories,” American Chemical Society Committee on
Chemical Safety: 1979.
S. “Handbook of Quality Control in Wastewater Laboratories,” U.S. Environmental Protection
Agency, Environmental Monitoring and SupportLaboratory, Cincinnati, OH,: EPA-60014-79-019,
March 1979.
6. ‘Standard Practice for Sampling Water” (ASTM Annual Book of Standards), American Society
for Testing and Materials, Philadelphia, Pennsylvania: 76, 1980.
7. ‘Methods 330.4 and 330.5 for Total Residual Chlorine,” U.S. Environmental Protection Agency,
Environmental Monitoring and Support Laboratory, Cincinnati, OH: EPA-600/4-70-020, March
1979.
8. “Narrative for SAS 1019,” Pacific Analytical, Inc.: September 1989. Available from the U.S.
Environmental Protection Agency Sample Control Center, 300 N. Lee St., Alexandria, VA 22314
(703-557-5040).
2. “Working with Carcinogens,” DHEW, PHS, CDC, NIOSH, Publication 77-206, (Aug 1977).
3. “OSHA Safety and Health Standards, General Industry” OSHA 2206, 29 CFR 1910 (Jan 1976).
4. “Safety in Academic Chemistry Laboratories,” ACS Committee on Chemical Safety (1979).
5. “Handbook of Quality Control in Wastewater Laboratories,” USEPA, EMSL, Cincinnati, OH
45268, EPA-600/4-79-019 (March 1979).
6. “Standard Practice for Sampling Water,” ASTM Annual Book of Standards, ASTM, Philadelphia,
PA, 76 (1980).
7. “Methods 330.4 and 330.5 for Total Residual Chlorine,” USEPA, EMSL, Cincinnati, OH 45268,
EPA 600/4-70-020 (March 1979).
783
-------
Method 1660
8. Narrative for SAS 1097M, Analytical Technologies Inc., September 1991. Available from the
USEPA Sample Control Center, 300 N Lee St,Alexandria, VA 22314 (703-557-5040).
784
-------
Method 1660
Table 1. Pyrethrins and Pyrethroids Determined by High Performance Liquid
Chromatography with Ultra-Violet Absorption Detector
Compound CA S Registry
Allethrin (Pynamin) 583-79-2
Cyfluthrin (Baythroid) 68359-37-5
Fenvalerate (Pydrin) 5 1630-58-1
Cis-permethrin 61949-76-6
Trans-perrnethrin 61949-77-7
Pyrethrin l 121-21-1
Pyrethrin II 121-29-9
Resmethrin 10453-86-8
Sumithrin (phenothrin) 26002-80-2
Tetramethrin 7696-12-0
Table 2. High Performance Liquid Chromatography of Pyrethrins and
Pyrethroids
Retention Time Minimum LeveP MDL 2
Compound (mm) (pg/U (pg/U
Pyrethrin l l 17.48 33 19
Tetramethrin 18.98 50 16
Allethrin 19.27 50 16
Pyrethrin I 20.89 31 22
Cyfluthrin 21.84 50 22
Resmethrin 22.07 50 22
Fenvalerate 22.68 25 10 est.
CIT-permethrin’ 22.98 50 20 est.
Sumithrin 23.47 50 25
C/T-permethrin’ 23.56 50 20 est.
This is a minimum level at which the analytical system shall give recognizable signals and
acceptable calibration points.
2. 40 CFR Part 136, Appendix B. Column system and conditions: two 150 mm long by 4.6 mm
ID 300 Angstrom C18 columns connected in series preceded by a 30 mm long by 4.6 mm ID 300
Angstrom C18 guard column. Column temperature 30°C. Solvent flow rate 1.5 mL/min.
Gradient: linear from 70% waterl30% acetonitrile at injection to 100% acetonitrile in 25 minutes.
3. Elution order of cis/trans isomers not known.
785
-------
Method 1660
Table 3. Concentration of Calibration Solutions
Allethrin
Resinethrin
C/T_permethrin*
CfF permethrin*
Table 4. Acceptance Criteria for Performance Tests
Pyrethroids
for Pyrethrins and
Solution Concentration (pg/mU
Compound
Calibration Solution I
Cyftuthrin
Fenvalerate
P_ I
Pyrethrin l l
Sumithrin
Tetramethrin
Calibration Solution 2
Low
Median
High
0.50
4.00
40.0
0.25
2.00
20.0
0.31
2.50
25.0
0.33
2.65
26.5
0.50
4.00
40.0
0.50
4.00
40.0
0.50
4.00
40.0
0.50
4.00
40.0
0.50
4.00
40.0
Acceptance Criteria
h7ithIPmcislon and
Calbiat Ion
Recovery
Accuracy
Ver ification 1
Ongoing Accuracy
(pg/Li
(pg/Li
R (pg/Li
Spike
Level
(pg t)
400
400
200
400
400
265
250
400
400
400
Mlethrin
C-
Fenvalerate
Cfl permedffh
C!Fpermethrin
P-I
Pyrethrinfl
Ream
Sundthr i a
Tetrame*hrin
S
90
125
35
75
75
70
60
125
140
90
x
160 - 520
110 - 610
120 - 260
230 - 530
230 - 530
60-320
110 - 330
43 - 510
46-570
170 - 530
3.5 - 4.6
3.0 - 5.2
1.6 - 2.4
3.0 - 4.6
3.0 - 4.6
2.2 - 2.8
2.0 - 3.5
2.3 - 5.2
3.5 - 4.7
1.5 - 6.1
150 - 530
94 - 630
62 - 314
220 - 540
210 - 540
77 - 330
100 - 340
25 - 520
25 - 590
150 - 550
1. VerIfied at the level of the median standard inTable 3.
2. First of two perinethrin peaks
3. Second of two permedirmn peaks
786
-------
Method 1660
1
—
0
C
a)
C j
0 )
I-
C
a)
E
I-
0 5.0 10.0 15.0 20.0 25.0
Retention Time (minutes)
52-002-85
Figure 1. Chromatogram of Pyrethrins and Pyrethroids
787
-------
Method 1661
The Determination of
Bromoxynil in Municipal and
Industrial Waste water
-------
790
-------
Method 1661
The Determination of Bromoxynil in
Municipal and Industrial Wastewater
1. SCOPE AND APPLICATION
1.1 This method covers the determination of bromoxynil in waste by direct aqueous injection high
performance liquid chromatography (HPLC) with an ultraviolet detector (UV).
1.2 This method is designed to meet the monitoring requirements of the U.S. Environmental
Protection Agency under the Clean Water Act at 40 CFR Part 455. Any modification of this
method beyond those expressly permitted shall be considered a major modification subject to
application and approval of alternative test procedures under 40 CFR 136.4 and 136.5.
1.3 When this method is applied to the analysis of unfamiliar samples, compound identity must be
supported by at least one additional qualitative technique. This method lists a second UV
wavelength that can be used to confirm measurements made with the primary wavelength.
1.4 The detection limit of this method is usually dependent on the level of interferences rather than
instrumental limitations. The limit in Table 1 typifies the minimum quantity that can be detected
with no interferences present.
1.5 This method is for use by or under the supervision of analysts experienced in the use of a high
performance liquid chromatograph and interpretation of liquid chromatographic data. Each
laboratory that uses this method must demonstrate the ability to generate acceptable results using
the procedure in Section 8.2.
2. SUMMARY OF METHOD
2.1 A 40- 1 iL aliquot of sample is injected into the HPLC. Chromatographic conditions are described
that permit the separation and measurement of bromoxynil by reverse-phase C18 colunm HPLC
with a multiple-wavelength UV detector.
2.2 Identification of bromoxynil is performed by comparing the retention time of the chromatograph
peak with that of an authentic standard. Compound identity is confirmed when the retention times
agree, and when the response at a second wavelength agrees with the response at the primary
wavelength.
2.3 Quantitative analysis is performed using an authentic standard of bromoxynil to produce a
calibration factor or calibration curve, and using the calibration data to determine the
concentration of bromoxynil in the sample.
2.4 Quality is assured through reproducible calibration and testing of the HPLC system.
3. CONTAMINATION AND INTERFERENCES
3.1 Solvents, reagents, glassware, and other sample-processing hardware may yield artifacts and/or
elevated baselines causing misinterpretation of chromatograms. All materials used in the analysis
791
-------
Method 1661
shall be demonstrated to be free from interferences under the conditions of analysis by running
method blanks as described in Section .8.4.
3.2 Glassware and, where possible, reagents are cleaned by rinsing with solvent and baking at 450°C
for 1 hour minimum in a muffle furnace or kiln. Some thermally stable materials may not be
eliminated by this treatment and thorough rinsing with acetone and pesticide-quality acetonitrile
may be required.
3.3 Specific selection of reagents and purification of solvents by distillation in all-glass systems may
be required.
3.4 interferences coextracted from samples will vary considerably from source to source, depending
on the diversity of the site being sampled.
4. SAFETY
4.1. The toxicity or carcinogenicity of each compound or reagent used in this method has not been
precisely determined; however, each chemical compound should be treated as a potential health
has d . Exposure to these compounds should be reduced to the lowest possible level. The
laboratory is responsible for m*intaining a current awareness file of OSHA regulations regarding
the safe hindling of the chemicals specified in this method. A reference file of material handling
sheets should also be made available to all personnel involved in these analyses. Additional
inform t1on on laboratory safety can be found in References 1 to 3.
4.2 Unknnwn samples may cOnt2in high concentrations of volatile toxic compounds. Sample
containers should be opened in a hood and handled with gloves that will prevent exposure.
5. APPAR4nls AND MA TERIALS
NOTE: Brand nwnes, suppliers, and part numbers are for illustrative purposes only. No
endorsement is inq,lied. Equiwdent peifrnnance may be achieved usIng apparatus and
materials other than those specified here, but demonstration of equivalent performance
meeting requlsiements of this method Is the responsibilhly of the laboratoiy.
5.1 Sampling equipment for discrete or composite sampling.
5.1.1 Sample bottle amber glass, 40-mL minimum, with screw-cap. If amber bottles are not
available, samples shall be protected from light.
5.1.2 Bottle caps: Threaded to fit sample bottles. Caps shall be lined with FFFE.
5.1.3 Cleaning.
5.1.3.1 Bottles are detergent water washed, then rinsed with solvent rinsed or baked
at 450°C for 1 hour minimum before use.
5.1.3.2 Liners are detergent-water washed, then rinsed with reagent water and
solvent, and baked at approximately 200°C for 1 hour minimum prior to use.
5.1.4 Compositing equipment Automatic or manual compositing system incorporating glass
containers cleaned per bottle cleaning procedure above. Sample containers are kept at
0 in 4°C during sampling. Glass or PTFE tubing only shall be used. If the sampler uses
792
-------
Method 1661
a peristaltic pump, a minimum length of compressible silicone rubber tubing may be used
in the pump only. Before use, the tubing shall be thoroughly rinsed with methanol,
followed by repeated rinsings with reagent water to minimize sample contamination. An
integrating flow meter is used to collect proportional composite samples.
5.2 Equipment for glassware cleaning.
5.2.1 Laboratory sink with overhead fume hood.
5.2.2 Kiln: Capable of reaching 450°C within 2 hours and holding 450°C within ± 10°C, with
temperature controller and safety switch (Cress Manufacturing Co, Sante Fe Springs,
CA, B31H or X31TS, or equivalent).
5.3 pH meter, with combination glass electrode.
5.4 Sample vials: Amber glass, 10- to 15-mL with PTFE-lined screw- or crimp-cap, to fit HPLC
autosampler.
Balance: analytical, capable of weighing 0.1 mg.
Miscellaneous glassware.
Pipettes, glass, volumetric, 1.00-, 5.00-, and 10.0-mL.
Pipettes, glass, Pasteur, 150 mm long by 5 mm ID (Fisher Scientific 13-678-6A, or
equivalent).
5.6.3 Volumetric flasks, 10.0-, 25.0-, and 50.0-mL.
5.7 High performance liquid chromatograph (HPLC): Analytical system complete with pumps,
sample injector, column oven, and multiple-wavelength ultra-violet (UV) detector.
5.7.1 Pumping system: Capable of isocratic operation (Waters 600E, or equivalent).
5.7.2 Sample injector: Capable of automated injection of up to 30 samples (Waters 700, or
equivalent).
5.7.3 Column oven: Capable of operation at room ambient to 50°C (Waters TCM, or
equivalent).
5.7.4 Column: 150 mm long by 4.6 mm ID 300 Angstrom C18 column (Vydac 201 TP5415,
or equivalent), operated at the conditions shown in Table 1.
5.7.5 Detector: UV operated at 255 and 280 nm (Waters 490E, or equivalent).
5.8 Data system.
5.8.1 Data acquisition: The data system shall collect and record LC peak areas and retention
times on magnetic media.
5.8.2 Calibration: The data system shall be used to calculate and maintain lists of calibration
factors (response divided by concentration) and multi-point calibration curves.
Computations of relative standard deviation (coefficient of variation) are used to test
calibration linearity.
5.8.3 Data processing: The data system shall be used to search, locate, identify, and quantify
the compounds of interest in each analysis. Displays of chromatograms are required to
verify results.
5.8.4 Statistics on initial (Section 8.2) and ongoing (Section 12.5) performance shall be
computed and maintained.
5.5
5.6
5.6.1
5.6.2
793
-------
Method 1661
6. REAGENTS AND STANDARDS
6.1 Sample preservation: Sodium thiosulfate (ACS), granular.
6.2 pH Adjustment.
6.2.1 Sodium hydroxide (iON): Dissolve 40 g NaOH in 100 mL reagent water.
6.2.2 Sulfuric acid (1 + 1): Reagent grade, 6N in reagent water. Slowly add 50 mL H 2 S0 4
(specific gravity 1.84) to 50 mL reagent water.
6.3 Solvents: Methaenl; pesticide-quality, lot-certified to be free of interferences.
6.4 Reagent water: HPLC grade water in which the compounds of interest and interfering compounds
are IK)t detected by this m hod.
6.5 Standard; Purchased as a solution with certification as to purity, concentration, and authenticity,
or prepared from materials of klx)wn purity and composition. If compound purity is 96% or
greater, the weight may be used without correction to compute the concentration of the standard.
When not being used, standards are stored in the dark at - 20°C to -10°C in screw-capped vials
with FWE-Iined lids. A mark is placed on the vial at the level of the solution so that solvent
evaporation loss can be detected . The vials are brought to room temperature prior to use.
6.6 PreparatIon of stock solutions: Prepare in water per the steps below. Observe the safety
precations in Section 4.
6.6.1 Dissolve an appropriate anx*int of assayed reference material in solvent. For example,
weigh 10 mg bromoxynil in a 100-mL ground-glass stoppered volumetric flask and fill
to the mark with water. After the bromoxynil is completely dissolved, transfer the
solution to a 150-mL vial with PTFE-Iined cap.
6.6.2 Stock solutsons should be checked for signs of degradation prior to the preparation of
calibration or peiforinance test standards.
6.6.3 Stock solutions shall be replaced after 6 months, or sooner if comparison with quality
control check standards indicates a th2nge in concentration.
6.7 Secondary mixtures: Using stock solutions (Section 6.6), prepare mixtures for calibration and
calibratlofl verlficatloà (Sections 7.3 and 12.4), for initial and ongoing precision and recovery
(Sections 8.2 and 12.5), and for spiking into the sample matrix (Section 8.3).
6.7.1 Calth Ion solutions: Prepare in water at the concentrations given in Table 2. The low
level solution is used fix calibration verification (Section 12.4)
6.7.2 Precision and recovery staádard and matrix spike solution: Prepare in water at a
concentration of 10 pglmL.
6.8 Stthilk ofaalutions: All stmvt d solutions (Sections 6.6 through 6.7) shall be analyzed within
48 Of preparMion and on a monthly basis thereafter for signs of degradation. Standards will
• & ai acoepishle if the peak area resn inii within ± 15% of the area obtained in the initial analysis
of the st n4ard .
7. SETWi AND CALERA liON
7.1 Configure the HPLC system described in Sections 5.7 through 5.8 and establish the operating
coalItions InTable 1.
7 ,94
-------
Method 1661
7.2 Attainment of minimum level: Determine that the minimum level in Table I is met at each
wavelength.
7.3 Calibration.
7.3.1 Inject 40 L of each calibration solution (Table 2) into the HPLC system, beginning
with the lowest concentration and proceeding to the highest. Compute and store, as a
function of the concentration injected, the retention time and the peak area of Bromoxynil
each wavelength (primary and confirmatory).
7.3.2 Calibration factor (ratio of area to amount injected).
7.3.2.1 Compute the coefficient of variation (relative standard deviation) of the
calibration factor over the calibration range at each wavelength.
7.3.2.2 Linearity: if the calibration factor is constant (C < 15%) over the
calibration range, an average calibration factor may be used; otherwise, the
complete calibration curve (area vs. amount) shall be used.
8. QUALITY CONTROL
8.1 Each laboratory that uses this method is required to operate a formal quality control program. 4
The minimum requirements of this program consist of an initial demonstration of laboratory
capability, an ongoing analysis of standards and blanks as tests of continued performance, and
analysis of spiked samples to assess accuracy. Laboratory performance is compared to established
performance criteria to determine if the results of analyses meet the performance characteristics
of the method.
8.1.1 The analyst shall make an initial demonstration of the ability to generate acceptable
accuracy and precision with this method. This ability is established as described in
Section 8.2.
8.1.2 The analyst is permitted to modify this method to improve separations or lower the costs
of measurements, provided all performance requirements are met. Each time a
modification is made to the method or a cleanup procedure is added, the analyst is
required to repeat the procedure in Section 8.2 to demonstrate method performance. If
minimum level will be affected by the modification, the analyst is required to repeat
demonstration of the minimum level (Section 7.2).
8.1.3 The laboratory shall, on an ongoing basis, demonstrate through calibration verification
and the analysis of the precision and recovery standard (Section 6.7.2) that the analysis
system is in control. These procedures are described in Sections 12.1, 12.4, and 12.5.
8.1.4 The laboratory shall maintain records to define the quality of data that is generated.
Development of accuracy statements is described in Section 8.3.
8.1.5 Analyses of blanks are required to demonstrate freedom from contamination. The
procedures and criteria for analysis of a blank are described in Section 8.4.
8.2 Initial precision and recovery: To establish the ability to generate acceptable precision and
accuracy, the analyst shall perform the following operations.
795
-------
Method 1661_
8.2.1 Analyze one set of four l0-mL aliquots of reagent water spiked with 100 pL of the
precision and recovery standard (Section 6.7.2) according to the procedure in Sections 10
and 11.
8.2.2 Using the results of the set of four analyses, compute the average recovery (X) and the
standard deviation of recovery (s) for bromoxynil.
8.2.3 Compare s and X with the corresponding limit for initial precision and recovery in
Table 1. If s and X meet the acceptance criteria, system performance is acceptable and
analysis of blanks and samples may begin. If, however, s exceeds the precision limit or
X falls outside the range for accuracy, system performance is unacceptable. In this case,
correct the problem and repeat the test.
8.3 Method acairacy The aboratory shall spike (matrix spike) at least 10% of the samples from a
given site type (e.g., influest to treatment, treated effluent, produced water). If only one sample
from * given site type is analyzed, a separate aliquot of that sample shall be spiked.
8.3.1 The concentration of the matrix spike shall be determined as follows.
8 .3.1.1 If, as in compliance monitoring, the concentration of bromoxynil in the
sample is being checked against a regulatory concentration limit, the matrix
spike shall be at that limit or at I to 5 times higher than the background
concentration determined in Section 8.3.2, whichever concentration is larger.
8.3.1.2 If the concentration is not being checked against a regulatory limit, the matrix
spike shall be at the level of the precision and recovery standard
(Section 6.7.2) or at 1 to 5 times higher than the background concentration,
whichever concentration is larger.
8.3.1.3 If it is impractical to determine the background concentration before spiking
(e.g., maximum holding times will be exceeded), the matrix spike
concentration shall be the regulatory concentration limit, if any; otherwise,
at the the level of the precision and recovery standard (Section 6.7.2) or
at 1 to 5 times the expected background concentration concentration,
whichever is larger.
8.3.2 Analyze one sample aliquot to determine the background concentration (B) of
bromoxynlL If necessary, prepare a stand2rd solution appropriate to produce a level in
the s n 4e 1 to 5 times the background concentration. Spike a second sample aliquot
with the st2iidard solution and analyze it to determine the concentration alter spiking (A)
of each analyte. Calculate the percent recovery (P):
E aUcn I
p = 100(4-B)
i *ere
T=
Th,ewthieq 1 the ike
796
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Method 1661
8.3.3 Compare the percent recovery of bromoxynil with the corresponding QC acceptance
criteria in Table 1. If it fails the acceptance criteria for recovery, the sample may be
complex and must be diluted and reanalyzed per Section 15.
8.3.4 As part of the QC program for the laboratory, method accuracy for samples shall be
assessed and records shall be maintained. After the analysis of five spiked samples of a
given matrix type (e.g., influent to treatment, treated effluent,produced water) in which
the the recovery test (Section 8.3.3) is passed, compute the average percent recovery (P)
and the standard deviation of the percent recovery ( )• Express the accuracy assessment
as a percent recovery interval from P - 2s, to P + 2s for each matrix. For example, if
P = 90% and s = 10% for five analyses of wastewater, the accuracy interval is
expressed as 70 to 110%. Update the accuracy assessment in each matrix on a regular
basis (e.g., after each 5 to 10 new accuracy measurements).
8.4 Blanks: reagent water blanks are analyzed to demonstrate freedom from contamination.
8.4.1 Analyze a reagent water blank with each sample batch (samples started through the
extraction process on the same 8-hour shift, to a maximum of 20 samples). Analyze the
blank immediately after analysis of the precision and recovery standard (Section 12.5) to
demonstrate freedom from contamination.
8.4.2 If any compound or any potentially interfering compound is found in an aqueous blank
at greater than 100 jigIL (assuming the same calibration factor as bromoxynil for
interfering compounds), analysis of samples is halted until the source of contamination
is eliminated and a blank shows no evidence of contamination at this level.
8.5 The specifications contained in this method can be met if the apparatus used is calibrated properly,
then maintained in a calibrated state. The standards used for calibration (Section 7), calibration
verification (Section 12.4), and for initial (Section 8.2) and ongoing (Section 12.5) precision and
recovery should be identical, so that the most precise results will be obtained. The HPLC
instrument will provide the most reproducible results if dedicated to the settings and conditions
required for the analyses of the analytes given in this method.
8.6 Depending on specific program requirements, field replicates and field spikes may be required to
assess the precision and accuracy of the sampling and sample transporting techniques.
9. SAMPLE COLLECTiON, PRESERVATiON, AND HANDUNG
9.1 Collect samples in glass containers following conventional sampling practices, 5 except that the
bottle shall not be prerinsed with sample before collection. Aqueous samples which flow freely
are collected in refrigerated bottles using automatic sampling equipment.
9.2 Maintain samples at 0 to 4°C from the time of collection until extraction. If the samples will not
be extracted within 72 hours of collection, adjust the sample to a pH of 3.0 to 7.0 using sodium
hydroxide or hydrochloric acid solution. Record the volume used. If residual chlorine is present
in aqueous samples, add 80 mg sodium thiosulfate per liter of water. EPA Methods 330.4
and 330.5 may be used to measure residual chlorine. 6
9.3 Begin sample extraction within 7 days of collection, and analyze all extracts within 44) days of
extraction.
797
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Method 1661
10. PREPARATiON OF SAMPLE AND QC AUQUOTS
10.1 Mix sample thoroughly.
10.2 Pour approximately 10 mL of sample into a clean HPLC autosampler vial. If a matrix spike is
to be prepared, pour 10.0 mL into a second clean vial.
10.3 Foreachsainpleorsamplebatch(to a maximum of 20) to be analyzed in the same 8-hour shift,
place two 10.0-mL aliquots of reagent water (Section 6.4) in clean auto-sampler vials. One
reagent water aliquot serves as the blank.
10.4 SpIke 100 1 cL of the precision and recovery standard (Section 6.7.2) into the remaining reagent
w ealicjuO(.
10.5 Spike the s n le designated as the matrix spike at the level directed in Section 8.3.
10.6 Analyze the sMr4)le and QC aliquots per Section 11.
11. N N PE RMANCE LIQWD CHROMATOGRAPHY
Table 1 summarizes the recommended operating conditions for the HPLC system. Included in
this Table Is the retention times for bromoxynil achieved under these conditions. An example of
the separation achieved by the column system is shown in Figure 1.
11.1 Calibrate the system as described in SectIon 7.
11.2 Set the Injection volume on the auto-sampler to inject 40 L of all standards, blanks, and samples.
11.3 Set the data system or HPLC control to begin data collection upon injection and to stop data
collection after bromoxynil is expected to elute.
12 SYSTEM AND LABORATORY PERFORMANCE
12.1 At the beginning of each 8-hour shift during which analyses are performed, HPLC system
perfbruzai e and àalibratlon are verified at both wavelengths. For these tests, analysis of the
calibration verification standard (Section 6.7.1) shall be used to verify all performance criteria.
Mjuim and/or recalibration (per Section 7) shall be performed until all performance criteria
are met. Caily after all performance criteria are met may samples, blanks, and precision and
recovery standards be analyzed.
12.2 Retention thn .
12.2.1 The M.ohite retention thne of bromoxynil shall be no less than 2.5 minutes.
122.2 The al solute retention time of the bromoxynil peak maximum shall be within ±15
seconds of the average of the retention times in the initial calibration (Section 7.3.1).
12.3 OC resolution: Resolution is acceptable if the peak width at half-height of bronioxynil
Is less than 15 seconds.
12.4 CalibratIon verification.
12.4.1 Inject the calibration verification standard (Section 6.7.1).
12.4.2 Com o the concn ration of bromoxynil based on the calibration factor or calibration
airve (Section 7.3).
798
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Method 1661
12.4.3 Compare this concentration with the limits for calibration verification in Table 1. If
calibration is verified, system performance is acceptable and analysis of blanks and
samples may begin. If, however, the recovery falls outside the calibration verification
range, system performance is unacceptable. In this case, correct the problem and repeat
the test, or recalibrate (Section 7).
12.5 Ongoing precision and recovery.
12.5.1 Analyze the precision and recovery standard prepared with each sample batch
(Section 10.3).
12.5.2 Compute the recovery of bromoxynil.
12.5.3 Compare the recovery with the limit for ongoing recovery in Table 1. If the recovery
meets the acceptance criteria, the analytical process is in control and analysis of blanks
and samples may proceed. If, however, the recovery falls outside the acceptable range,
these processes are not in control. In this event, correct the problem and repeat the
ongoing precision and recovery test.
12.5.4 Add results which pass the specifications in Section 12.5.3 to initial and previous ongoing
data. Update QC charts to form a graphic representation of continued laboratory
performance. Develop a statement of laboratory data quality for each analyte by
calculating the average percent recovery (R) and the standard deviation of percent
recovery Sr. Express the accuracy as a rôcovery interval from R - 2s to R + 25r. For
example, if R = 95% and Sr = 5%, the accuracy is 85 to 105%.
13. QLIAUTA TIVE DETERMINA TFON
13.1 Qualititative determination is accomplished by comparison of data from analysis of a sample or
blank with data from analysis of the shift standard (Section 12.1), and with data stored in the
retention-time and calibration libraries (Section 7.3.1). Identification is confirmed when retention-
time and amounts agree per the criteria below.
13.2 Establish a retention-time window ±15 seconds on either side of the mean retention-time in the
calibration data (Section 7.3.1).
13.3 If a peak from the analysis of a sample or blank is within this window (as defined in Section 13.2)
at the primary wavelength (280 nm), it is considered tentatively identified. A tentatively identified
compound is confirmed when (1) the retention time of the peak maximum at the confirmatory
wavelength (255 nni) is within ±2 seconds of the retention-time of the peak maximum at the
primary wavelength, and (2) the computed amounts (Section 14) on each system (primary and
confirmatory) agree within a factor of 2.
14. QUANT1TA T1VE DETERMINA TJON
14.1 Using the HPLC data system, compute the concentration of the analyte detected in the sample (in
milgram per liter) using the calibration factor or calibration curve (Section 7.3.2).
14.2 If the concentration of any analyte exceeds the calibration range of the system, the sample is
diluted by a factor of 10, and a 4OjiL aliquot of the diluted extract is analyzed.
799
-------
Method 1661
14.3 Report results for bromoxynil found in all standards, blanks, and samples to three significant
figures. Results for samples that have been diluted are reported at the least dilute level at which
the concentration is in the calibration range.
15. ANAL Y&’S OF CoMPLEx SAMPLES
15.1 Some samples may contain high levels (>1000 pg/L) of bromoxynil or of interfering compounds
and/or polymeric materials. Some sani 1 Aes may overload the HPLC column and/or detector. In
these instances, the .c nI)le is diluted by a factor of 10 and reanalyzed (Section 14.2).
15.2 Recovery of matrix spikes: in most samples, matrix spike recoveries will be similar to those from
reag * wMer. If the matrix spike recovery is outside the range specified in Table 1, the sample
is diluted by a factor of 10, resplked, and rein2lyzed. If the matrix spike recovery is still outside
the range, the med d does ect work on the sample being analyzed and the result may not be
— for regulatory compliance purposes.
16. METhOD PEFFORMANCE
18.1 Development of this method i detailed hi Reference 7.
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Method 1661
References
1. “Carcinogens: Working with Carcinogens.” Department of Health, Education, and Welfare;
Public Health Service; Center for Disease Control; National Institute for Occupational Health and
Safety: Publication 77-206, Aug 1977.
2. “OSHA Safety and Health Standards, General Industry” (29 CFR 1910). Occupational Safety and
Health Administration: Jan 1976.
3. “Safety in Academic Chemistry Laboratories,” American Chemical Society Committee on
Chemical Safety: 1979.
4. “Handbook of Quality Control in Wastewater Laboratories,” U.S. Environmental Protection
Agency, Environmental Monitoring and SupportLaboratory, Cincinnati, OH,: EPA-600/4-79-019,
March 1979.
5. “Standard Practice for Sampling Water” (ASTM Annual Book of Standards), American Society
for Testing and Materials, Philadelphia, Pennsylvania: 76, 1980.
6. “Methods 330.4 and 330.5 for Total Residual Chlorine,” U.S. Environmental Protection Agency,
Environmental Monitoring and Support Laboratory, Cincinnati, OH: EPA-600/4-70-020, March
1979.
7. “Narrative for SAS 1019,” Pacific Analytical, Inc.: September 1989. Available from the U.S.
Environmental Protection Agency Sample Control Center, 300 N. Lee St., Alexandria, VA 22314
(703-557-5040).
801
-------
Meffiod 1661
Table 1. HPLC Data and Method Acceptance Criteria for Bromoxynil *
Acceptance
Minimum Level
100 pgIL
I
Method Detection limit
20 pg/L
2
Calibration Verification (Section 12.4)
86 to 128 pg/L
3
Initial Precision and Recovery (Section 8.2)
Precision (st Ivd deviation, a]
Recovery (m ’n; XJ
28 igIL
74 to 130 jig/L
3
Ongoing Precision and Recovery (Section 12.5)
Matrix Spike Recovery (Section 8.3.3)
Bromoxynil Rm ke-time
fl to 132 igIL
68 to 129%
2.62 minutes
4
(3,5ydroxybenzonitrlle CAS 1689-84-5)
Netes:
I. This Is a mflmuw level at whlth the analytical system shall give recognizable signals and
accq*able calibration points.
2. Estimat 40 ( R Part 136, Appendix B.
3. Test ccnc atiMlos 100 tg/L.
4. C1 n and conditions: 150 mm long by 4.6 mm ID 300 Angstrom C18 column Column
ten ,er*ure 30°C. Solvers flow rate 0.5 mLlmin. Isocratic at 50% methanol in water.
Table 2. Concentration of Bromoxynil Calibration Solutions
Note
L•vel Can
Low 100
Median 6(X)
High 3,000
802
-------
M.thod 1661
Figure 1. Chromatogram of Bromoxynhl
0.0
0.5 1.0 1.5 2.0 2.5
Retention Time (minutes)
3.0 3.5
A52M0246
803
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A PPENDIX
Methods EV-024
and EV-025
Analytical Procedures for
Determining Total Tin and
Triorganotin in Waste water
Provided by
ATOCHEM North America
-------
Methods EV-024 and EV-025
Analytical Procedures for Determining Total Tin
and Triorganotin in Waste water
EV-024
SAFETY
Wear rubber gloves and glasses with side shields. Follow standard laboratory safety procedures. Any
special safety notes are included in the procedure body.
2. PROCEDURE
2.1 Prepare the AA & HGA 500 with the appropriate instrumental operating conditions and keyboard
entries:
2.1.1 AA2380
2.1.1.1 Turnpoweron.
2.1.1.2 Open H 2 0 drain exit valve and 1120 inlet valve (% open).
2.1.1.3 Install Sn hollow cathode element and regular, uncoated tube.
2.1.1.4 Check to see that all control knobs are in extreme counter-clockwise position.
2.1.1.5 Using lamp control knob, set lamp/energy to 30 mA.
2.1.1.6 Set slit nm to 0.7 ALT and wavelength to 286.3.
2.1.1.7 Adjust wavelength and lamp alignment (by turning signal to set up position,
turning gain control knob clockwise until about 35 registers on lamp/energy
display, then adjusting beat and lamp for maximum gain).
2.1.1.8 Position control knobs as follows:
Signal - conc
Mode - PkHT
Recorder - TCL
BG Correction - AA KN
2.1.1.9 Set integration time for 7 seconds.
2.1.1.10 Turn on inert gas supply (argon).
2.1.2 HGA 500
2.1.2.1 Turn power on.
2.1.2.2 Program keyboard with following entries.
807
-------
Appendix: IND-Ol
Step
1 2 3 4
Temp °C 100 700 2500 50
RampTime(s) 15 15 0 0
Hold Time (s) 15 15 7 5
Rec. x x x
Read x
Mini Flow, Argon x x
mL/min 50 50
Stop flow x
2.2 Secure appropriate amount (usually 25 to 150 mL depending on source of sample) directly into
previously cleaned (see comments) 500-mL screw-top bottle.
2.3 Add 10 mL hydrobromic acid to each sample. Shake well then let stand for 10 minutes.
2.4 Add 50 mL (or more, also dependent upon source of sample) 0.05% tropolone in toluene (F-i’)
solution. Shake for 10 minutes. Allow to separate into two layers.
2.5 If separation is sufficient (i.e., little emulsion), run 5 L on HGA-500 AA 2380 after
standardization. (See Step 6); if separation unsatisfactory, add 10 mL more of T-T solution, shake
10 minutes and let stand to separate into two layers.
2.6 To standardize:
2.6.1 Inject 5 1 tL of 0.05 ppm standard inorganic Sn into the graphite furnace with an
Eppendorf pipette, equipped with a clean, disposable tip.
2.6.1 Png*ge program cycle and record peak height.
2.7 Repeat star4ard until numbers are reproducible and close in range and average.
2.8 Qieck cleanliness of furnace by engaging program cycle without sample until baseline returns.
2.9 Obtain S pL directly from upper phase of each sample from Step 5 using the Eppendorf pipette
technique with a clean tip for each sample and run through cycle at least three times and average
results.
2.10 Run 5 giL of blank (F-T solution) three times and average.
a CALCULATiONS
7 = Real conc. x ml T-T solution used
— w ume
Conc - Average ABS normalized to 5 pL xfactor
Ezan le
1. Norma lizeallaverageto5pL;
e.g., 25 giL sample average = 30.4 + 5 = 6.08
2. Determine or of standard.
808
-------
Appendix: EV-024 and EV-025
Concentration of standard in ppm
Average ABS (nonnalize to 5 L)
Example:
Avg ABS of 0.5 ppm was .115
et) 0.5/.115 = 4.348 = Factor
Example:
Avg ABS of sample is 0.146. Dilution factor is lOx. Sample volume is 150 mL.
T-T solution volume Is 50 mL. (Use previous example as factor for standard).
( 4.38)(50X0.146)(lOx ) = 2.116 ppm Total un
4. COMMENTS
4.1 Mark original level of sample in sample bottle so that volume may be determined.
4.2 Amount of sample extract introduced into the graphite furnace may vary depending upon actual
concentration.
4.3 Suitable signals for analysis are obtained in the lox expansion mode.
4.4 All sample bottles and glassware used must be scrupulously cleaned with concentrated HCI, by
soaking for several hours or overnight, and then rinsed several times with distilled water.
4.5 Label bottles with magic marker for sample name, date sample discharged, sample volume, and
T-T volume.
809
-------
A: 1
EV-025
1. SAFETY
1.1 Wear tubber gloves aM glasses with side shields. Follow standaid laboratory safety procedures.
Any qecial safety es are included in tbe procedure body.
2. FWJCEDWIE
2.1 Prqare AA aM tote tin san les as ec1 d hi EV4324.
2.2 Usir de * vials, etia 2 at ftijài phe of u al tin sMIlple.
2.3 Add etpal wt of pcepaied 3% NaOH solution to san le in vial. Label vial with .sample name
le mkar.
2.4 Shake ftr 10 diuii •
2.5 biJect S at on HGA 500, AA 23 0 after tan’ !ardis*Ion hdo graphite furnace with an Eppendorf
p ett , e ilpped with a clean dlqx*thle tip.
2.6 Engage program cycle and recoid peak height
2.7 Rqs* a Ee dese In tags and r rOducible, and average.
a Cis L4 flows
Calad*Ions are the s as In ppm total tla.’
Fe**%T-T Voi*eX4vg A&S%Dth ion )
S.iq’ie V 1wr
O5ppm stamlaid with Avg. 0.115 ABS
( 0.5 ) 4.348 Faacr
ppm Th Th Avg ABS of s-’ 4 . Is 0.123. Dibdlon factor is none, but 25 pL was used.
S Ie Is 150 at ad T-T solution volume is 50 at.
( 4348X50)(0.123) + - o.o R m
810
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